184: LS Midterm #2
In eukaryotic cells, three distinct RNA polymerases are responsible for transcribing RNAs with different functions.
RNA polymerase I (Pol I), RNA polymerase II (Pol II), and RNA polymerase III (Pol III)
The chemistry and mechanism of RNA synthesis closely resemble those of DNA synthesis. How?
RNA polymerase extends an RNA strand by adding ribonucleotide units to the 3′-hydroxyl end building RNA in the 5′→3′ direction.
Early experiment demonstrating DNA-dependent RNA synthesis by an RNA polymerase.
RNA polymerizing activity was partially purified from E. coli and analyzed for RNA synthesis using a template DNA of sequence (AT)n and various rNTP substrates. Reactions took place in the presence of nonradioactive (unlabeled) rNTPs and one radiolabeled rNTP, as noted on the graph. Incorporation of radioactively labeled rNTP substrates was measured.
read length for reversible terminator sequencing
Read lengths are shorter for this method, typically 100 to 200 nucleotides per cluster, although refinements are ongoing.
how to renature a denatured DNA?
Reducing the temperature will often allow a denatured DNA to renature.
Sanger sequencing method:
Refers to two methods developed by Frederick Sanger, (1) for determining the sequence of a polypeptide (2) for determining the base sequence of a DNA molecule.
Function of Base Methylation in DNA
Regulating Gene Expression
wild-type recA gene from an altered recA gene via automated sequencer
Results from an automated sequencer showing sequences from the wild-type recA gene (top) and from an altered recA gene (recA K72R, bottom) with the triplet (codon) at position 72 changed from AAA to CGC, specifying an Arg (R) instead of a Lys (K) residue. Here, the nucleotide colors reflect the dyes actually used in the method, and thus deviate from the standard nucleotide colors used in other figures.
DNA is a double helix
Rosalind Franklin, Raymond Gosling, and Maurice Wilkins were measuring x-ray diffraction of DNA fibers generated by drawing them from solution using a glass rod. The diffraction patterns reflected the symmetry of the DNA molecules in the fibers, thereby providing an important clue to the molecules' overall arrangement. By examining the diffraction pattern from fibers oriented perpendicular to the x-ray beam, investigators could deduce the helical symmetry of the molecules inside. These data were interpreted in the light of Chargaff's rules, which state that in a given DNA sample, the fraction of A equals the fraction of T, and the fraction of C equals the fraction of G. Possible models of the DNA in the fiber were produced, and their calculated diffraction patterns were compared with the experimentally derived patterns.
Additional condensation of cellular DNA is facilitated by
SMC proteins
SMC structure
SMC proteins are generally dimeric, forming a V-shaped complex linked through the hinge domains. Thus the dimeric SMC complex contains two head domains and two ATP-binding sites. Although many details of SMC protein function have yet to be elucidated, the head-head association between the two subunits seems to be critical.
SMC proteins have five distinct domains
SMC proteins have five distinct domains The amino-terminal (N) and carboxyl-terminal (C) domains each contain part of an ATP-hydrolytic site, and they are connected by two regions of α-helical structure joined to a hinge domain.
Summary of the structure of SMC proteins.
SMC proteins have five domains.
Part 6i - DNA Sequence Analysis
Sample B - Stool Sample (See Samples section for more info) After the sequence information has been gathered from all the reaction tubes, the computer builds the actual sequence by matching together different pieces. You now have the 16S rDNA sequence for this bacterial species, which can be compared with all other known 16S rDNA sequences for identification. Learn about the science behind sequence matching. There are many sequence databases in existence. Some databases are sold commercially as part of an identification kit. In this example, we will use the GenBank public database available through the National Library of Medicine. The matching algorithm is known as BLAST (Basic Local Alignment Search Tool). A direct match of the DNA sequence determines the exact bacterial species you have found. When the DNA sequence is not an exact match but a close match to another found in the sequence database, you need to assess whether it is a new species or a variation of an existing one.
deamination
Several nucleotide bases undergo deamination, a spontaneous loss of their exocyclic amino groups.
cohesins
SMC proteins that link sister chromatids immediately after chromosomal replication and keep them together as the chromosomes condense to metaphase.
Condensation of cellular chromosomes is facilitated by
SMC proteins, including cohesins and condensins
Cohesins are made up of
SMC1-SMC3 pairs
condensins consist of
SMC2-SMC4 pairs. The SMC2-SMC4 complex is called a condensin.
how can STR loci be analyzed?
STR loci can be analyzed by PCR.
example of 454 sequencing with dGTP
Similarly, when dGTP is added, flashes occur at a different set of clusters, marking those as clusters where G is the next nucleotide added to the sequence.
examples of modifications
Some of the first examples were discovered in ribosomal and transfer RNAs. In some cases, modifications involve the addition of a functional group to an existing nucleotide in the sequence. For example, a methyl group can be added to the 2′ hydroxyl of ribose, thereby blocking its ability to form a hydrogen bond. In bacteria, some C residues of tRNAs are modified to N4-acetylcytidine in a process thought to contribute to the accuracy of protein synthesis. In other cases, the base itself is changed, or its linkage to the ribose—the glycosidic bond—is altered. For instance, inosine, 4-thiouridine, and pseudouridine are relatively common in tRNAs and rRNAs.
affect of larger tags
Some of the larger tags, such as maltose-binding protein, provide added stability and solubility, allowing the purification of cloned proteins that are otherwise inactive due to improper folding or insolubility.
site-directed mutagenesis.
Specific amino acids may be replaced individually by site-directed mutagenesis.
Part 2 - PCR Amplification; Step 1
Step 1: Add Master Mix To prepare the polymerase chain reaction (PCR), we will add the PCR Master Mix solution to our sample DNA. At the same time as the test reaction, we will prepare negative and positive control reactions. Instead of the sample DNA, the positive control reaction contains positive control DNA (the solution of 16S rDNA in the green-capped bottle) while the negative control reaction contains sterile deionized water. Both reactions contain the PCR Master Mix solution.
Part 6ii - DNA Sequence Analysis Now follow these steps to identify your sample:
Step 1: Click here to see the data output from the sequencer. Select the all data in the window and copy (ctrl-c on the PC or cmd-c on the Mac). Step 2: Click here to go to the NCBI site to perform your search. (This will pop open a new window) Step 3: On the NCBI site's BLAST page, paste your data (ctrl-v on the PC or cmd-v on the Mac) in the box labeled "Enter accession number, gi, or FASTA sequence," in the "Enter Query Sequence" near the top of the page. In the "Choose Search Set" section, Database: "Others (nr etc.)" and "Nucleotide collection (nr/nt)" should already be selected. When you are ready, click on the blue "BLAST" button. (updated October 21, 2007) Step 4: Follow the instructions provided by the NCBI site to obtain your results. Step 5: Come back to this page (which should remain open) and identify the bacteria in the lab window to the left.
Part 2 - PCR Amplification Step 2: Run PCR
Step 2: Run PCR Once the reaction tubes are loaded onto the thermocycler (the "PCR machine"), the automatic process of DNA replication starts. The machine used in this lab has readouts that describe what is happening: (from left to right: temperature, time remaining, cycle number, melt, anneal, and extend) The temperature control is set up as follows: Initial incubation step: 95°C for 10 minutes 30 cycles of the following sequence of steps: Melt: 95°C 30 seconds Anneal: 60°C 30 seconds Extend: 72°C 45 seconds Final extension step: 72°C 10 minutes Final step: 4°C store at this temperature During each cycle, the first step (melt) is to separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95°C for 30 seconds. The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this temperature, the primers bind (anneal) to the single-stranded DNA. The final step (Extend) is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds. The three steps—the separation of the strands, annealing the primer to the template, and the synthesis of new strands—take less than two minutes. Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been duplicated. The cycle can be repeated 30 or more times, and each newly synthesized DNA piece acts as a new template. After 30 cycles, 1million copies of the initial DNA piece can be produced.
What helped define the fundamental properties of transcription?
Subsequent experiments using the purified E. coli RNA polymerase AND using bacteriophage RNA polymerases
What can electron microscopy visualize
Such denatured regions, or bubbles, can be visualized with electron microscopy
function of online databases with genuine sequences
Such programs provide unprecedented access to gene se- quence information. In turn, progress on this front is simplifying the process of cloning individual genes for more detailed analysis.
what are PCR primers attached to? why?
Suitable PCR primers (with an attached dye to aid in subsequent detection)
active underwinding of the DNA causes what?
Supercoiling is the direct result of structural strain caused by the active underwinding of the DNA—that is, the removal of helical turns relative to the most stable structure of B-form DNA.
initiation: synthesis of DNA, synthesis RNA, and synthesis of protein
(1) The first of three stages in the synthesis of DNA, in which the DNA polymerase binds to the origin of replication. (2) The first of three stages in the synthesis of RNA, in which the RNA polymerase binds to the promoter sequence on the DNA. (3) The first of three stages in the synthesis of a protein, in which the ribosome binds to the mRNA and initiator aminoacyl-tRNA
elongation: (RNA synthesis and protein synthesis)
(1) The second of three stages of RNA synthesis, in which ribonucleotides are added to the 3' end of the growing RNA molecule. (2) The second of three stages of protein synthesis, in which amino acids are added to the C-terminal end of the growing peptide chain.
Bacteria: topoisomerase II class? function?
(DNA gyrase) class: Type II function: introduces negative supercoils
The reason the two separated strands of DNA do not reanneal when the temperature is cool is because..?
(The reason the two separated strands of DNA do not reanneal is that there is a large excess of primers in the solution; therefore, it's more likely for the DNA strands to bind to the primers instead of to each other.)
454 sequencing
(the numbers refer to a code used during development of the technology and have no scientific meaning), uses a strategy called pyrosequencing in which the addition of nucleotides is detected by flashes of light (Figure 7-13).
how is the color associated with each band detected?
...and the color associated with each band is detected with a laser beam.
shows the steps in the reaction catalyzed by bacterial type I topoisomerases
1. A DNA molecule binds to the topoisomerase, and one DNA strand is cleaved 2. The enzyme changes conformation 3. the unbroken DNA strand moves through the break in the first strand 4. Finally, the DNA strand is ligated and released ATP is not used in this reaction. The enzyme promotes the formation of a less strained, more relaxed state by removing supercoils.
Example of a Bacterial Topoisomerase I reaction
1. Active site Tyr attacks a phosphodiester bonds one DNA strand, cleaving it and creating a covalent 5'-phosphtyrosyl protein-DNA linkage 2. Enzyme changes to open conformation 3. The unbroken DNA strand passes through the break in the first strand 4. Enzyme is closed conformation liberated 3'-OH attacks the 5'-phosphtyrosyl protein-DNA linkage to relegate the cleaved DNA strand
6 steps of 454 sequencing
1. The four dNTPs (unaltered) are pulsed onto the reacting surface one at a time in a repeating sequence. 2. The nucleotide solution is retained on the surface just long enough for DNA polymerase to add that nucleotide to any cluster where it is complementary to the next nucleotide in the template sequence. 3. Excess nucleotide is destroyed quickly by the enzyme apyrase before the next nucleotide pulse. 4. When a specific nucleotide is successfully added to the strands of a cluster, pyrophosphate is released as a byproduct. 5. Another enzyme in the solution bathing the surface is sulfurylase, which converts the pyrophosphate to ATP. 6. The appearance of ATP ultimately provides the signal that a nucleotide has been added to the DNA.
Introduction of negative DNA supercoils by DNA gyrase. (b) The mechanism of gyrase action.
1. The multi-subunit enzyme binds to a DNA segment 2. DNA is then wrapped around the complex in the N gate, stabilizing a positive node, involving two DNA segments (purple and light red) 3. The bound light red DNA is cleaved across both strands forming two 5'-phosphotyrosyl linkages to the enzyme (orange dots). The purple DNA segment passes through the break and exits the enzyme through the C gate 4. Enzyme relegates the cleaved strand and resets for another round of catalysis. The (+) node has been converted to a (-) node
general procedure of GST tag
1. the GST gene sequence is fused to a target gene 2. the fusion protein acquires the capacity to bind glutathione. 3. The fusion protein is expressed in a host organism such as a bacterium 4. and a crude extract is prepared.
Only one of the two DNA strands serves as template. This template DNA is copied in the
3′→5′ direction (antiparallel to the new RNA strand), just as in DNA replication.
polynucleotide synthesis proceeds in what direction?
3′→5′ direction.
Two next-generation sequencers
454 sequencing reversible terminator sequencing
The degree of underwinding in cellular DNAs generally falls in the range of
5% to 7%; that is, σ = −0.05 to −0.07.
After four cycles of PCR, how many NEW double stranded DNA copies were generated from the original template? (Note, count only double stranded newly synthesized DNA)
8 (half of the 16)
Southern blotting
A DNA hybridization procedure in which one or more specific DNA fragments are detected in a larger population by hybridization to a complementary, labeled nucleic acid probe.
DNA-dependent RNA polymerases require
A DNA template Mg2+ and all four rNTPs (ATP, GTP, UTP, and CTP) as substrates for the polymerization reaction.
how to separate His-tagged protein from other proteins
A chromatography matrix with immobilized Ni2+ can be used to quickly separate a His-tagged protein from other proteins in an extract.
expression vectors.
A cloning vector with the transcription and translation signals needed for the regulated expression of a cloned gene.
closed-circular DNA:
A continuous double-stranded DNA molecule with no free 3' or 5' ends. When these DNA molecules have no breaks in either strand, they are referred to as closed-circular DNAs.
does the ddNTP change for each reaction?
A different ddNTP is used in each reaction.
hybrid duplex:
A duplex experimentally reconstituted from single-stranded DNA (or RNA) from different sources.
APOBECs
A family of human proteins called APOBECs catalyze cytosine deamination in the viral genome during the initial round of replication by HIV. This hypermutation results in many nonviable viral particles, eventually destroying the coding capacity of the virus.
Cloned genes alterations?
A gene sequence can be changed, sequences deleted, or sequences added. All changes affect the protein or RNA product of the gene.
bacmid:
A large circular DNA that includes the entire baculovirus genome and sequences that allow replication of the bacmid in Escherichia coli; a baculovirus shuttle vector.
oligonucleotide-directed mutagenesis:
A method for creating a mutation in a cloned gene. Two short, complementary synthetic DNA strands, each with the desired base change, are annealed to opposite strands of the cloned gene within a suitable vector. The two annealed oligonucleotides prime DNA synthesis, creating two complementary strands with the mutation.
pyrosequencing:
A method for rapid DNA sequencing in which nucleotide additions are detected with flashes of light. detects the addition of nucleotides on the DNA to be sequenced by flashes of light.
ion torrent:
A method for rapid DNA sequencing.
single molecule real time (SMRT) sequencing:
A method for rapidly sequencing a long segment of a single strand of DNA.
reversible terminator sequencing:
A method used in many protocols for rapid DNA sequencing.
Northern blotting
A nucleic acid hybridization procedure in which one or more specific RNA fragments are detected in a larger population by hybridization to a complementary, labeled DNA probe.
Lys72? affect of changing Lys residue to an Arg
A particular amino acid residue in RecA (a 352-residue polypeptide), Lys72, is involved in ATP hydrolysis. By changing this Lys residue to an Arg, a variant of RecA protein is created that will bind, but not hydrolyze, ATP (Figure 7-20c). The engineering and purification of this variant RecA protein has facilitated research into the roles of ATP hydrolysis in the functioning of this protein.
tag
A peptide or protein that binds a simple, stable ligand with high affinity and specificity. Added sequences can produce protein products that include fused peptide segments, called tags. With the aid of these tags, the protein can be rapidly purified.
reverse transcriptase PCR (RT-PCR):
A polymerase chain reaction (PCR) protocol for amplifying an RNA sequence by first using reverse transcriptase to create a DNA copy.
quantitative PCR (qPCR):
A polymerase chain reaction (PCR) protocol that allows the simultaneous amplification and detection of a sequence through use of a fluorescent probe. See also polymerase chain reaction.
polymerase chain reaction (PCR):
A repetitive laboratory procedure that results in a geometric amplification of a specific DNA sequence.
contig:
A series of overlapping clones or a continuous sequence defining an uninterrupted section of a chromosome. In a successful genomic sequencing exercise, many contigs can extend over millions of base pairs.
site-directed mutagenesis:
A set of methods used to create specific alterations in the sequence of a gene.
short tandem repeat (STR):
A short (typically 3 to 6 bp) DNA sequence repeated multiple times in tandem at a particular location in a chromosome.
His tags
A shorter tag with widespread application consists of a simple sequence of six or more histidine residues. These histidine tags, or His tags, bind tightly and specifically to nickel ions.
If PCR analysis shows a mutation in the BRCA2 gene, that person will develop breast cancer??
A single mutation in the BRCA2 gene does not indicate that a person will develop breast cancer. Rather, it is indicative of an increased RISK of that person's developing breast cancer at some point in their life. Cancer rarely results from a single mutation. Typically, another mutation in a different gene, in conjunction with the first mutation, is required to induce cancer. This principle is called the "two-hit hypothesis."
sigma factor (σ):
A sixth subunit, binds transiently to the core and directs the enzyme to (the promoter) specific binding sites on the DNA. allows for the unwinding of DNA
probe
A specific gene's DNA or RNA sequence can be detected in the presence of many other sequences by hybridization with a probe a carefully chosen nucleic acid sequence complementary to the gene of interest. A labeled fragment of nucleic acid containing a nucleotide sequence complementary to a genomic sequence that one wishes to detect in a hybridization experiment.
whole-genome shotgun sequencing:
A strategy for sequencing a genome in which random segments of DNA are sequenced and the segments are ordered by the computerized identification of sequence overlaps.
plectonemic supercoiling:
A structure in a molecular polymer that has a net twisting of strands about each other in some simple and regular way.
Two approaches to site-directed mutagenesis.
A synthetic DNA segment replaces a fragment removed by a restriction endonuclease. A pair of synthetic and complementary oligonucleotides with a specific sequence change at one position are hybridized to a circular plasmid with a cloned copy of the gene to be altered.
protein structure and function changes
A variety of methods, based in large measure on techniques pioneered by Michael Smith and his colleagues in the late 1970s, are now used to enhance research on proteins by allowing investigators to make specific changes in the primary structure and examine the effects of these changes on the protein's folding, three-dimensional structure, and activity. This powerful approach to studying protein structure and function changes the amino acid sequence by altering the DNA sequence of the cloned gene.
The DNA sites where strand separation of DNA must occur in vivo during processes such as DNA replication and transcription are initiated are often rich in A=T base pairs.
A=T base pair
Whis is ATP hydrolyzed with SMC proteins
ATP is not hydrolyzed until the two heads come together.
role of additional proteins in relation to cohesin
Additional proteins, particularly proteins in the kleisin family such as SCC1, bridge the cohesin head units to form a ring
adenine, cytosine, and guanine (rate of methylation)? figure of modified nucleotides in DNA. most common postsynthetic modification?
Adenine and cytosine are methylated more frequently than guanine. Practice drawing uracil, guanine, thymine, uracil, and cytosine The most common postsynthetic modification to DNA is base methylation. 5-Methyldeoxycytidine occurs in the DNA of animals and higher plants; the other methylated bases shown here are produced by specific enzymes.
Terminal Tags Provide Handles for
Affinity Purification
what is the problem with Affinity chromatography
Affinity chromatography is one of the most efficient methods for purifying proteins. Unfortunately, many proteins do not bind a ligand that can be conveniently immobilized on a column matrix.
what happens once a PCR is complete? why?
After a PCR is complete, a portion of the product is usually run on a gel, to check if a DNA fragment of the predicted size was synthesized.
what happens to the signal after many PCR cycles?
After many PCR cycles, the signal reaches a plateau as one or more reaction components are exhausted.
After several cycles of PCR, what happens to the mutation-containing DNA? 3 points
After several cycles of PCR, the mutation-containing DNA predominates in the population and can be used to transform bacteria. Most of the transformed bacteria will have plasmids carrying the mutation. The template plasmid, usually isolated from wild-type E. coli, has a methylated A residue in every copy of the four-nucleotide palindrome GATC.
Why is a homodimers important in bacteria?
All bacteria have at least one SMC protein that functions as a homodimer to assist in compacting the genome
What happens to all four ddNTPs?
All four labeled ddNTPs are added together.
how do fragments move in the capillary gel?
All fragments of a given length migrate through the capillary gel together in a single band
Introduction to BRCA2 and the Polymerase Chain Reaction
All humans are born with two copies, or alleles, of the BRCA2 gene. If one of these alleles contains a harmful mutation, a person's lifetime risk of developing breast cancer is greatly increased. About 12% of women in the general population will develop breast cancer during their lifetime. But approximately 45% of women who inherit a harmful BRCA2 mutation will develop breast cancer by age 70. BRCA2 mutations are also linked to increased risk for ovarian and lung cancers.
What do DNA methylases (methyltransferases) use as a methyl group donor?
All known DNA methylases (methyltransferases) use S-adenosylmethionine as a methyl group donor
what happens to the other DNA in the sample?
All other DNA in the sample remains unamplified.
Also present in the medium is what enzyme???
Also present in the medium is the enzyme luciferase and its substrate molecule, luciferin
mutations
Alterations in DNA structure that produce permanent genetic changes are known as mutations. An inheritable change in the nucleotide sequence of a chromosome.
Alteration of Cloned Genes Produces
Altered Proteins
Do mRNAs have stable three-dimensional structures?
Although mRNAs were once thought to be spaghetti-like molecules, increasing evidence hints that they may have stable structures that contribute to biological function. For example, many mRNAs include long sequences that extend beyond the coding region of the gene and are critical for proper gene regulation. Specific proteins bind to these regions and probably recognize structures within them.
DNA genotyping (Allan Maxam and Walter Gilbert) vs Sanger method (Frederick Sanger)
Although the two methods are similar in approach, the Sanger method, also known as the dideoxy chain-termination method, has proved to be technically easier and is in more widespread use
plectonemic and solenoidal supercoiling similarities
Although their structures are very different, plectonemic and solenoidal supercoiling are two forms of negative supercoiling that can be adopted by the same segment of underwound DNA. The two forms are readily interconvertible.
bacteriophage T7
An alternative system uses a promoter and RNA polymerase found in a bacterial virus called bacteriophage T7. If the cloned gene is fused to a T7 promoter, it is transcribed not by the E. coli RNA polymerase but by the T7 RNA polymerase. The gene encoding this polymerase is separately cloned into the same cell in a construct that affords tight regulation (allowing controlled production of the T7 RNA polymerase). The polymerase is also very efficient and directs high levels of expression of most genes fused to the T7 promoter. This system has been used to express the RecA protein in bacterial cells (Figure 7-17).
topoisomerase:
An enzyme that catalyzes alterations in DNA topology, introducing and/or removing positive or negative supercoils in closed, circular duplex DNA. increase or decrease the extent of DNA underwinding by changing the linking number.
type II topoisomerase:
An enzyme that introduces positive or negative supercoils in closed, circular duplex DNA by cleaving both DNA strands, passing an intact segment of DNA through the break, then religating the broken ends. Type II topoisomerases change Lk in increments of 2.
type I topoisomerase:
An enzyme that introduces positive or negative supercoils in closed, circular duplex DNA by cleaving one of the two DNA strands, passing the intact strand through the break, and ligating the broken ends. Type I topoisomerases change Lk in increments of 1.
Analysis of multiple STR can produce what?
Analysis of multiple STR loci can yield a profile that is unique to an individual. This is typically done with a commercially available kit that includes PCR primers unique to each locus, linked to colored dyes to help distinguish the different PCR products.
sequence polymorphisms:
Any alterations in genomic sequence (base-pair changes, insertions, deletions, rearrangements) that help distinguish subsets of individuals in a population or distinguish one species from another.
how are topoisomerases crucial to cancer???
As a consequence, they are important drug targets for the treatment of bacterial infections and cancer. Inactivating mutations in genes that encode key cellular topoisomerases, the enzymes responsible for the degree of supercoiling in cells, often result in severe growth deficiencies or cell death.
what happens in the beginning of chromosome segregation?
As chromosome segregation begins, the cohesin tethers are removed by enzymes known as separases.
what is DNA genotyping based on
As first described by English geneticist Alec Jeffreys in 1985, the method is based on sequence polymorphisms, slight sequence differences among individuals—1 in every 1,000 bp, on average.
As the PCR reaction proceeds, what happens to the amount of the targeted DNA segment? what about the fluorescent signal?
As the PCR reaction proceeds, the amount of the targeted DNA segment increases exponentially, and the fluorescent signal also increases exponentially as the oligonucleotide probes anneal to the amplified segments.
how does the fusion protein become immobilized
As the crude extract percolates through this matrix, the fusion protein becomes immobilized by binding to the glutathione.
roles of condensins in the eukaryotic cell cycle.
At the onset of mitosis, condensins bind and maintain the chromatids in a condensed state. During anaphase, the enzyme separase removes the cohesin links. Once the chromatids separate, condensins begin to unload and the daughter chromosomes return to the uncondensed state. condensins provide a general structural scaffold for chromosome condensation.
explain the structure and length of plectonemic supercoiling
At the superhelical densities normally encountered in cells, the length of the supercoil axis (the axis about which the supercoils turn), including branches, is about 40% of the length of the DNA. This type of supercoiling is referred to as plectonemic supercoiling
common hosts for protein expression?
Bacteria, especially E. coli, remain the most common hosts for protein expression. The regulatory sequences that govern gene expression in E. coli and many other bacteria can be harnessed to express cloned proteins at high levels.
Figures of NAD NADP+ FAD adoMet
Be able to draw the,
The effects of replication and transcription on DNA supercoiling.
Because DNA is a double-helical structure, strand separation leads to added stress and supercoiling if the DNA is constrained (not free to rotate) ahead of the strand separation. The general effect can be illustrated by twisting two strands of a rubber band about each other to form a double helix. If one end is constrained, separating the two strands at the other end will lead to twisting.
why are two different STR lengths produced?
Because each human inherits one chromosome of each chromosome pair from each parent, the STR lengths on the two chromosomes are often different, generating two different STR lengths from one individual.
What kind of DNA fragments migrate faster? short or long?
Because shorter DNA fragments migrate faster,
Why is the phosphodiester bond not simply hydrolyzed
Because this would entail loss of a high-energy bond, and an activation step would then be required to promote the subsequent ligation.
Why is underfunding important to the enzymes of DNA replication and transcription?
Because underwinding facilitates strand separation which must bring about strand separation as part of the enzyme's function
What does blocking groups on each fluorescently labeled nucleotide prevent?
Blocking groups on each fluorescently labeled nucleotide prevent multiple nucleotides from being added per cycle.
How does supercoiling affect replication and transcription?
Both processes require a separation of DNA strands, which is complicated by the helical interwinding of the strands (Figure 9-8).
what became more powerful than fingerprints?
But with the advent of recombinant DNA technology, a much more powerful tool became available: DNA genotyping (also called DNA fingerprinting or DNA profiling).
how can the design of primers use for PCR alter the amplified segment?
By careful design of the primers used for PCR, the amplified segment can be altered by the inclusion, at each end, of additional DNA not present in the chromosome that is being targeted.
what is CT?
CT is the cycle number at which the threshold is first surpassed.
How to yield an estimate of its base composition?
Careful determination of the melting point of a DNA specimen, under fixed conditions of pH and ionic strength, can yield an estimate of its base composition.
after DNA synthesis what residues are often methylated?
Certain A and C residues in DNA are often enzymatically methylated
enzymatically methylation and when?
Certain nucleotide bases in DNA molecules are enzymatically methylated, usually after DNA synthesis is complete.
examples of changes to a gene
Changes can be introduced into a gene that involve far more than one base pair. Large parts of a gene can be deleted by cutting out a segment with restriction endonucleases and ligating the remaining portions to form a smaller gene. Parts of two different genes can be ligated to create new combinations; the product of such a fused gene is called a fusion protein. Researchers have ingenious methods to bring about virtually any genetic alteration in vitro. After reintroducing the altered DNA into the cell, they can investigate the consequences of the alteration.
Chromosomal DNAs vs rotation
Chromosomal DNAs are typically complexed with many proteins that inhibit free rotation.
why do Cloned genes look for in promoters when linked to them?
Cloned genes must be linked to promoters that can direct high-level expression in yeast. For example, the yeast GAL1 and GAL10 genes are under cellular regulation such that they are expressed when yeast cells are grown in media with galactose but shut down when the cells are grown in media with glucose. Thus, if a heterologous gene is expressed using the same regulatory sequences, the expression of that gene can be controlled simply by choosing an appropriate medium for cell growth.
purpose of Cloning techniques
Cloning techniques can be used not only to overproduce proteins but to produce protein products subtly altered from their native forms.
roles of cohesins in the eukaryotic cell cycle.
Cohesins are loaded onto the chromosomes during G1 tie the sister chromatids together during replication. Cohesins tether the sister chromatid products of DNA replication
The simplest examples of RNA polymerases
Consist of one polypeptide chain, such as the phage T7 and Sp6 RNA polymerases.
Electrophoresis separates DNA based on
Correct! Charge is one characteristic that is used to separate DNA because DNA is negatively charged and will migrate toward the positive cathode in electrophoresis. Size is the other characteristic that is used to separate DNA because smaller fragments tend to migrate farther.
methylation is most common at what sequence?
CpG sequences, producing methyl-CpG symmetrically on both strands of the DNA.
Promoting cruciform structures by DNA underwinding.
Cruciforms can form at palindromic sequences, but they seldom occur in relaxed DNA because linear DNA accommodates more paired bases than the cruciform structure. DNA underwinding facilitates the partial strand separation required for promoting cruciform formation at appropriate sequences.
The structure of the ddNTP pentose ring is most similar to that of the pentose ring of
DNA The pentose ring of RNA has two hydroxyl groups, the pentose ring of DNA has one hydroxyl group, and the pentose ring of dideoxynucleotide (ddNTP) has no hydroxyl groups. Therefore, the ddNTP pentose ring is most similar to the pentose ring of DNA. Keep in mind that during a Sanger sequencing reaction, you are sequencing a DNA molecule, so it would make sense that the ddNTPs are most similar to the dNTPs of DNA.
DNA Underwinding Is Defined
DNA Underwinding Is Defined by the Topological Linking Number
Linking number and no topological property
DNA can have a topological property such as a linking number only if both strands are intact. If there is a break in either strand, the strands can, in principle, be unraveled and separated; in this case, no topological bond exists and Lk is undefined
what happens to DNA over time?
DNA degrades slowly over time
DNA genotyping has been used to
DNA genotyping has been used to both convict and acquit suspects, and to establish paternity with an extraordinary degree of certainty.
For a PCR reaction, two primers are used because:
DNA is double stranded DNA polymerase synthesizes DNA in the 5'→3' direction. The forward primer anneals to one template strand, the reverse primer anneals to the other template strand, and both initiate DNA polymerase reactions in the correct direction.
Supercoiling results when
DNA is subject to some form of structural strain.
Solenoidal supercoils are formed when
DNA is wrapped around the nucleosomes that make up eukaryotic chromatin. Similarly, in bacteria, the tight wrapping of DNA around a variety of DNA-binding proteins gives rise to solenoidal supercoils.
Topoisomerases have functions specific to
DNA metabolism, such as unlinking catenated bacterial DNA after replication or relaxing supercoils formed by unwinding during replication and transcription.
DNA molecules in solution denature when
DNA molecules in solution denature when they are heated slowly.
Step by Step Mechanism for RNA synthesis. What group is the nucleophile? What group is the electrophile? LG?
The 3′-hydroxyl group makes a nucleophilic attack on the α phosphate of the incoming rNTP, with the concomitant release of pyrophosphate.
Where does the DNA polymerase bind too?
DNA polymerase cannot bind to the template DNA strand alone. It binds to the DNA-primer hybrid.
PCR procedure relies on what enzymes?
DNA polymerases.
DNA sequences in a typical E. coli expression vector
DNA sequences in a typical E. coli expression vector. The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end of the gene encoding the N-terminus of the protein positioned closest to the promoter.
"next-gen" sequencing
DNA sequencing technologies continue to evolve. These advances have been made possible by methods sometimes referred to as next-generation, or "next-gen" sequencing. The sequencing strategy is sometimes similar to and sometimes quite different from that used in the Sanger method. Innovations have allowed a miniaturization of the procedure, a massive increase in scale, and a corresponding decrease in cost.
DNA supercoiling/DNA underwinding influences what part of DNA
DNA supercoiling—or, more specifically, DNA underwinding—is a precisely regulated process that influences many aspects of DNA metabolism.
DNA synthesis reaction steps
DNA synthesis involves a reaction between the 3′-hydroxyl group of the primer dNTP and the phosphate group of an incoming dNTP.
DNA synthesis reaction/mechanism
DNA synthesis involves a reaction between the oxygen in the 3'-hydroxyl group at the 3' end of the growing strand and the α-phosphate group of an incoming dNTP that is complementary to the next free nucleotide in the template strand.
supercoiled DNA
DNA that twists upon itself because it is underwound or overwound (and thereby strained) relative to B-form DNA.
In the laboratory, condensins bind ?
DNA to create positive supercoils
The underwinding and relaxation of DNA are catalyzed by
DNA topoisomerases, enzymes that break one or both DNA strands to allow a topological change, and then religate them.
DNA topoisomers that migrate faster in the gel?
DNA topoisomers that are more supercoiled are more compact
dna underwinding and cruciform formation
DNA underwinding helps maintain the required strand separation in regions where palindromic sequences allow cruciform formation
topoisomerases
DNA underwinding is catalyzed by enzymes called topoisomerases, and the degree of DNA underwinding is highly regulated in every cell.
What does this decreased activity cause?
Decreased activity of one of these enzymes (e.g., by mutation) causes growth deficiencies, which can be partially relieved by compensating mutations in the gene encoding the other enzyme.
Determination of which locus corresponds to which signal depends on what??
Determination of which locus corresponds to which signal depends on the color of the fluorescent dye attached to the primers used in the process and on the size range in which the signal appears (the size range can be controlled by which sequences—those closer to or more distant from the STR—are targeted by the designed PCR primers).
topoisomers
Different forms of a covalently closed, circular DNA molecule that differ only in their linking number. Two forms of a circular DNA that differ only in a topological property such as linking number are referred to as topoisomers.
Discontinuous deformations
Discontinuous deformations involve DNA strand breakage.
double helix unwinding
Disruption of both the hydrogen bonding between paired bases and the base stacking causes the double helix to unwind, forming two single strands that are completely separate from each other
How and why does the double helix unwind?
Disruption of both the hydrogen bonding between paired bases and the base stacking causes the double helix to unwind, forming two single strands that are completely separate from each other along the entire (or partial) length of the molecule.
requirements for Dpnl to cleave DNA at GATC
DpnI selectively cleaves DNA at the sequence GATC only if the A residue in one or both strands is methylated—that is, the enzyme breaks down only the template.
SMC polypeptide information
Each SMC polypeptide is folded so that the two coiled-coil domains wrap around each other and the N and C domains come together to form a complete ATP-binding site. Two polypeptides are linked at the hinge region to form the dimeric V-shaped SMC molecule.
Be able to describe the six successive cycles from one very small part of an Illumina sequencing run.
Each colored spot represents the location of an immobilized DNA oligonucleotide affixed to the surface of the flow cell. The circled clusters represent the same spot on the surface over successive cycles and give the sequences indicated. Data are automatically recorded and analyzed digitally.
relation between each cycle of heating, cooling, and replication and the number of DNA segment?
Each cycle doubles the amount of the DNA segment, so the concentration of this DNA grows exponentially.
prototype human genome sequence
Each difference from the prototype human genome sequence (the first one obtained) occurs in some fraction of the human population; every person has some differences from this prototype.
DNS segment sequencing and introduction of the bead
Each individual segment of template DNA to be sequenced is attached to a tiny DNA capture bead then amplified on the bead by PCR. Each bead is immersed in an emulsion and placed in a tiny (∼ 29 μm) well on a picotiter plate. Analyzing the figure: Circles represent the same cluster over multiple cycles. In this case, reading the top (or bottom) circle from left to right across each row gives the sequence for that cluster.
Modified nucleotides in RNA and figure
Enzyme-catalyzed RNA base modifications are common in tRNA and rRNA, although the function of such alterations is not always clear. The presence of N4-acetylcytidine in bacterial tRNAs may enhance protein synthesis.
function of type II enzymes
Eukaryotic type II topoisomerases cannot underwind DNA (introduce negative supercoils), but they can relax both positive and negative supercoils. They function in all aspects of eukaryotic DNA metabolism, resolving a range of topological problems that occur during replication, transcription, and repair. They play an especially important role in the condensation of chromosomes into highly structured chromatin.
How is supercoiling an intrinsic property of DNA tertiary structure?
Even when extracted and purified, many circular DNA molecules remain highly supercoiled, even in the absence of protein and other cellular components. This indicates that supercoiling is an intrinsic property of DNA tertiary structure, as opposed to an incidental result of spatial constriction.
What does the resulting strained state represent?
Every cell actively underwinds its DNA with the aid of enzymes, and the resulting strained state represents a form of stored energy.
Many Different Systems Are Used to
Express Recombinant Proteins
aging, carcinogenesis (initiation of cancer formation), and mutations
Extensive evidence suggests an intimate link between the accumulation of mutations in an individual organism and the processes of aging and carcinogenesis.
Week 5: Transcription of DNA into RNA.
The DNA duplex opens to allow a complementary RNA copy to be made from one DNA strand (the template). Synthesis proceeds in the 5′→3′ direction in the growing RNA strand.
GyrA
The DNA exits through a domain in GyrA called the C-gate. A separate domain of GyrA binds DNA and promotes DNA wrapping.
Sanger sequencing can only be used on DNA for which the sequence is unknown. True or False
FALSE Sanger sequencing will work on almost any sequence of DNA, regardless of whether the template sequence is known. While it is possible to sequence DNA for which the sequence is known, it is typically not done because the information obtained (i.e., the sequence of the DNA) is already known.
Step 3 of Gyrase Reaction?
The DNA is bound such that a positive node (a crossover of two DNA segments that cross in the sense of a positive supercoil) is created in the active site. Active-site Tyr residues bind ATP and cleave both strands of one of the DNA segments
during these topological transactions....
The DNA is never released from the enzyme so uncontrolled relaxation of the DNA does not occur.
Step 1 of synthesis of nucleic acids
First, a nucleotide is attached to the support, a glass or polystyrene bead, through its 3′-hydroxyl group
Underwinding accomplishes what two things?
First, cells maintain DNA in an underwound state in part to promote its compaction by coiling. Second, underwinding facilitates strand separation and enzymatic access to the encoded information;
A genomic sequence is generated in several steps: (5 steps)
First, the genomic DNA is broken at random locations by shearing to generate fragments that are a few hundred base pairs long. Synthetic oligonucleotides are ligated to the ends of all the fragments, providing a known point of reference on every DNA molecule. The individual fragments are then immobilized on a solid surface, and each is amplified by PCR (see Figure 7-9). The solid surface is part of a channel that allows liquid solutions to flow over the samples. The result is a solid surface just a few centimeters wide, with millions of attached DNA clusters, each cluster containing multiple copies of a single DNA sequence derived from a random genomic DNA fragment.
Example of Northern blot analysis of RNA isolated from various human tissues.
For each sample, approximately 10 μg of total RNA was separated on a 1.2% agarose-formaldehyde gel, transferred to a membrane, and hybridized to a 32P-labeled probe—an mRNA for human platelet endothelial cell adhesion molecule (PECAM-1). The same blot was also probed with a cDNA (complementary DNA, a DNA copy of an mRNA sequence) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to control for the amount of material in each lane. (GAPDH mRNA is used as a control because it is found in all tissues, in almost equal amounts.) Note the differences in PECAM-1 RNA levels detected in the different tissues; two bands are observed for PECAM-1 in each lane because there are two distinct forms of the mRNA for this gene.
amplified of sequences in RNA is an example of specialized adaptions of PCR. How can this occur?
For example, sequences in RNA can be amplified if reverse transcriptase is used in the first PCR cycle (see Figure 7-8). After the DNA strand is made using the RNA as a template, the remaining cycles can be carried out with a DNA polymerase by normal PCR protocols.
deamination of cytosine, uracil, adenine, guanine (in regards to rate!)
For example, under typical cellular conditions, deamination of cytosine (in DNA) to uracil occurs in about 1 of every 107 C residues in 24 hours. This corresponds to about 100 spontaneous deamination events per day, on average, in a mammalian cell. Deamination of adenine and guanine occurs at about 1/100th this rate.
example of 454 sequencing with dCTP
For example, when dCTP is added to the solution, flashes occur only at clusters where G is the next base in the template and C is the next nucleotide to be added to the growing DNA chain. If there is a string of two, three, or four G residues in the template, a similar number of C residues are added to the growing strand in one cycle. This is recorded as a "flash" amplitude at that cluster that is two, three, or four times greater than when only one C residue is added.
deep sequencing
For some applications, the amounts of genomic DNA to be sequenced are increased so that sequencing depth is increased to 100 × or even 1,000 ×. This approach, sometimes called deep sequencing can help determine whether a mutation is present in a subset of an organism's cells or whether other genomic variations are present. Deep sequencing is helpful in the characterization of genomic sequences in cancerous tumors, where the genome is highly unstable and changes frequently as the tumor grows. Extensive genomic sequencing designed to produce multiple (sometimes hundredfold or greater) coverage of all targeted sequences; used to detect sequence variants within a population.
Oligonucleotide synthesis is very useful
For techniques such as Southern and Northern blotting and for the polymerase chain reaction (PCR) and DNA sequencing
Part 5 - DNA Sequencing
From the last step, you have 12 tubes that contain the final PCR product, a mix of DNA pieces of variable length. All DNA pieces in each tube start with the same primer but end with a different nucleotide tagged with a fluorescent marker (different color for each nucleotide A, T, G, C). What remains to be done is to separate the individual DNA pieces and identify the end nucleotide. This is done by using an automatic sequencer that performs gel electrophoresis on the DNA in each tube. Gel electrophoresis is a method to separate molecules based on differences in size. The sequencer used in this lab has a thin capillary tube attached at one end to a syringe mechanism that contains a buffer solution. The tube is filled with the buffer solution and the other end inserted into one of the tubes containing the DNA pieces. Then, an electric current is applied so that the end of the tube in contact with the DNA has a negative charge and the syringe end a positive charge. Since DNA molecules are negatively charged, they start to move through the tube toward the positively charged syringe end, with the smaller pieces moving faster than the larger ones. Near the syringe end, the capillary tube passes through a laser beam that excites the fluorescent markers, and optical detectors detect the color of the fluorescence. We can assume that a complete set of DNA pieces, all differing in size by exactly one nucleotide, were generated in the previous step. The smallest piece of DNA that has a fluorescent tag attached to it is the primer. This DNA fragment will travel faster than the other ones. By reading the color (in our example, red), we determine that the first nucleotide beyond the primer sequence is thymidine (T). The next smallest piece of DNA will fluoresce with the color (green) representing the next nucleotide in the sequence (A) and so on (see the animation). By reading the sequence of nucleotides based on their fluorescence as the DNA pieces pass through the laser beam, the sequence of the DNA can be reconstructed. The sequencer automatically flushes out the buffer from the tube, moves the tray, and runs the electrophoresis again, repeating the program until all 12 tubes have been examined. The resulting sets of sequences are collated by a computer program to build the complete sequence of the 16S rRNA gene.
Eukaryotic Topoisomerases Have Specialized what?
Functions in DNA Metabolism
where are well-characterized promoter and its regulatory elements positioned? and why?
Generally, a well-characterized promoter and its regulatory elements are positioned near several unique restriction sites for cloning, so that genes inserted at the restriction sites will be expressed from the regulated promoter elements
With specialized adaptations of PCR, investigators can ...
Genes or other DNA segments can be amplified by the polymerase chain reaction. With specialized adaptations of PCR, investigators can amplify sequences in RNA and quantify the levels of particular RNA molecules in a cell.
genetic analysis of heterologous protein shows what type of results? what bad thing can happen? economical?
Genetic analysis or direct demonstration of heterologous protein expression then identifies animals in which the gene transfer has been successful, and these animals are bred. Posttranslational protein modifications are not always carried out correctly for proteins expressed in this way, but protein production can be economical once a line of protein-expressing animals is established.
because PCR methods are sensitive, what can potentially be a serious issue and what is done to control it?
Given the extreme sensitivity of PCR methods, contamination of samples is a serious issue. In many applications, including forensic and ancient DNA tests, controls must be run to make sure the amplified DNA is not derived from the researcher or from contaminating bacteria.
Some of the same problems that accompany protein expression in bacteria also occur with yeast.
Heterologous proteins may not fold properly, yeast may lack the enzymes needed to modify the proteins to their active forms, or the expression of proteins may be made difficult by certain features of the gene sequence.
Solutions of carefully isolated, double-stranded DNA are
Highly viscous at pH 7.0 and room temperature (25°C)
host efficiency and the expression of eukaryotic genes
However, because S. cerevisiae is a eukaryote, the expression of eukaryotic genes (especially yeast genes) is sometimes more efficient in this host than in bacteria. Folding and modification of the products may also be more accurate than for proteins expressed in bacteria.
big affect of tags, even small, on proteins. elaborate? and how to avoid this?
However, even very small tags can affect the properties of the proteins they are attached to, thereby influencing the study results. Even if the tag is removed by a protease, one or a few extra amino acid residues can remain behind on the target protein, which may or may not affect the protein's activity. The types of experiments to be carried out, and the results obtained from them, should always be evaluated with the aid of well-designed controls to assess any effect of a tag on protein function.
How does RNA-modifying enzymes give cells a selective advantage over organisms that do not modify their RNA?
However, genetic experiments in which an RNA-modifying enzyme is mutated or deleted from an organism suggest that these enzymes give cells a subtle but important selective advantage over organisms that do not modify their RNA. Some evidence supports the hypothesis that RNA modifications stabilize RNA structures and help RNAs interact with proteins in the cell.
"Leaky" lactose system
However, regulation in the lactose system is "leaky": it is not turned off completely when lactose is absent—a potential problem if the product of the cloned gene is toxic to the host cells. Transcription from the Lac promoter is also not efficient enough for some applications.
disadvantage of the growth of mammalian cells
However, the growth of mammalian cells in tissue culture is very expensive, and this technology is generally used to test the function of a protein in vivo rather than to produce a protein in large amounts.
Go deeper into why UV light decreased with H-bonding and base stacking (Hypochromic effect)
Hydrogen bonding and base stacking in the double helix limit the resonance of the aromatic rings of the bases, thereby decreasing UV light absorption.
quantitative PCR can reveal the increased representation of what kind of sequence.
If a DNA sequence is present in higher than usual amounts in a sample for example, certain genes may be amplified so that they are present in many copies in the cells that make up a cancerous tumor
what happens when appropriate restriction sites flank the sequence to be altered
If appropriate restriction sites flank the sequence to be altered, researchers can simply remove a DNA segment and replace it with a synthetic one identical to the original except for the desired change.
If A=T denatures can the DNA remain double stranded?
If denaturation conditions are carefully controlled, regions that are rich in A=T base pairs will specifically denature while most of the DNA remains double-stranded.
example of hybridizing DNA from different species
If double-stranded DNAs isolated from human cells and from mouse cells are completely denatured by heating, then mixed and kept at 25°C for many hours, much of the DNA will anneal. Most of the mouse DNA strands anneal with complementary mouse DNA strands to form mouse duplex DNA; similarly, most human DNA strands anneal with complementary human DNA strands. However, some strands of the mouse DNA will associate with human DNA to yield hybrid duplexes, in which segments of a mouse DNA strand form base-paired regions with segments of a human DNA strand
what happens if extra DNA sequences are included at the 5' end of the synthetic primers?
If extra DNA sequences are included at the 5′ end of the synthetic primers (e.g., the sequence specifying a restriction site, as shown here), those sequences are incorporated into the final product.
what can happen to the nonmutant template with Dpnl?
If necessary, the nonmutant template plasmid DNA can be selectively eliminated by cleavage with the restriction enzyme DpnI.
relaxed state-->adding strain
If one of these turns were removed, there would be (84 bp)/7 = 12.0 bp per turn, rather than the 10.5 found in B-DNA. This is a deviation from the most stable DNA form, and the molecule would be thermodynamically strained as a result. 9-10B
The linking number for a closed-circular DNA is always an integer. (+ vs -)
If the DNA strands are interwound in a right-handed helix, the linking number is positive (+) For strands interwound in a left-handed helix, the linking number is negative (−).
when is a closed-circular molecule relaxed rather than supercoiled
If the double helix of a closed-circular molecule has the features of B-form structure, with one turn of the double helix per 10.5 bp, it is relaxed rather than supercoiled
How are probes made?
If the protein product of a gene has been purified, probes can be designed and synthesized by working backward from the amino acid sequence, deducing the DNA sequence that would code for it. Or, researchers can often obtain the DNA sequence information necessary for creating a probe from sequence databases that detail the structure of millions of genes from a wide range of organisms.
one step verses two step renaturation process?
If the strands were completely separated, renaturation occurs in two steps. In the first step, which is relatively slow, complementary sequences in the two strands "find" each other by random collisions and form a short segment of double helix. The second step is much faster: the remaining unpaired bases successively come into register as base pairs, and the two strands "zipper" themselves together to form the double helix.
Under what condition can an underwound DNA revert spontaneously back to the relaxed state?
If there is a break in one strand of an isolated, protein-free circular DNA, free rotation at that point will cause the underwound DNA to revert spontaneously to the relaxed state.
minimal portion of DNA needed to undergo PCR
If we know the sequence of at least the end portions of a DNA segment we are interested in, we can hugely amplify the number of copies of that DNA segment with the polymerase chain reaction (PCR)
How can the linking number can be defined as what is regard to circles and surface
If we think of one of the circular strands as the boundary of a surface (such as the soap film on the loop of a bubble wand before you blow a bubble), the linking number can be defined as the number of times the second strand pierces this surface (Figure 9-12b).
Part 3ii - Purify PCR Product
If you are confident that the PCR worked, you can proceed to purifying the PCR product. Running a gel is actually one method of purification. Once the PCR product is in the gel, you can cut out the band corresponding to the PCR product and isolate the DNA from the gel. Nowadays, you can buy compact microfilters to filter the DNA from the PCR tube without running a gel. We will use such microconcentrator columns in our procedure: Insert the microconcentrator column of appropriate size into a collection tube. Add 400 µL of buffer to the column. Add the entire PCR content (~100 µL) to the column. Spin the column at 3,000 rpm in a fixed-angle centrifuge for 15 minutes. The PCR product should be trapped in the column while the collection tube should contain all the primers, nucleotides, and other small compounds that we no longer need. Remove the collection tube and discard it. Invert the column and attach it to a new collection tube. Add 50 µL of buffer to the inverted column. This step should loosen the DNA from the column into the collection tube. Spin the inverted column at 3,000 rpm for 2 minutes to collect the sample in the collection tube. Discard the column. The final collection tube should now have many pieces of 1,500bp-long 16S rDNA, with a very small amount of longer DNA strands (which are contaminants).
The second bacterial type II topoisomerase, DNA topoisomerase IV, has a very specialized function.
Immediately following replication, the circular daughter chromosomes of bacteria are topologically intertwined. Circular DNAs that are intertwined in this way are called catenanes. DNA topoisomerase IV unlinks the catenated daughter chromosomes, allowing their proper segregation at cell division.
In DNA, uracil is the product of
In DNA, uracil is the product of cytosine deamination
Vif
In HIV and related viruses, the viral protein Vif binds to APOBECs and triggers their degradation. Vif has therefore become an important antiviral target, because viruses lacking this protein are much less capable of establishing chronic infection in human cells.
RNA Molecules Are Often Site-Specifically Modified in what?
In Vivo: within an organism
In a DNA molecule, the progress of a DNA polymerase or RNA polymerase (as shown) along the DNA involves
In a DNA molecule, the progress of a DNA polymerase or RNA polymerase (as shown) along the DNA involves separation of the strands.
FIGURE 7-15: Sequence assembly: in debt explanation, read through, and understand
In a genomic sequence, each base pair of the genome is usually represented in many of the sequenced fragments, referred to as reads. Shown is a small part of the sequence of a new variant species of E. coli, with the reads generated by a 454 sequencer. The numbers at the top represent genomic base-pair positions, relative to an arbitrarily defined "0." The sequences all come from a particular long contig designated 356. The reads themselves are represented by horizontal arrows, with computer-assigned identifiers listed for each one at the left. DNA strand segments are sequenced at random, with sequences obtained from one strand (5′ to 3′, left to right) represented by solid arrows and sequences obtained from the other strand (5′ to 3′, right to left) represented by dashed arrows. The latter sequences are automatically reported as their complement when they are merged with the overall dataset. The "coverage threshold" at the top is a measure of sequence quality. The wider green bar indicates sequences that have been obtained enough times to generate high confidence in the results. The depth of the coverage line indicates how many times a given base pair appears in a sequenced read. The vertical blue shaded line indicates a part of the sequence that is highlighted by thin blue brackets in the sequence line at the bottom of the page. The "SNP statistics report" (inset) is a listing of positions where single nucleotide polymorphisms (SNPs; see Chapter 8) appear to be present in some of the reads. These putative SNPs are often checked by additional sequencing. They are indicated in the reads by thin, blue vertical slash marks within the horizontal lines for each read.
superhelical density (σ):
In a helical molecule such as DNA, the number of supercoils (superhelical turns) relative to the number of coils (turns) in the relaxed molecule. change in Lk/Lkinitial
PCR is carried out in the presence of ______ that emits _______ when _________ __________ is present?
In brief, the PCR is carried out in the presence of a probe that emits a fluorescent signal when the PCR product is present
In eukaryotic cells, what percentage of all C residues in DNA are methylated to form 5-methyldeoxycytidine
In eukaryotic cells, about 5% of all C residues in DNA are methylated to form 5-methyldeoxycytidine
G≡C base pairs vs A=T with melting point
In general, the higher the content of G≡C base pairs in the DNA, the higher its melting point. This is because G≡C base pairs, with three hydrogen bonds, require more heat energy to dissociate than do A=T base pairs.
How can the tag be removed from the purified fusion protein?
In some commercially available systems, the tag can be entirely or largely removed from the purified fusion protein by a protease that cleaves a sequence near the junction between the target protein and its tag.
dideoxynucleoside triphosphates (ddNTPs) function and structural explanation? EXAMPLE
In the Sanger sequencing reaction, nucleotide analogs called dideoxynucleoside triphosphates (ddNTPs) interrupt DNA synthesis because they lack the 3′-hydroxyl group needed for the next step (Figure 7-11b). For instance, the addition of ddCTP to an otherwise normal reaction system causes some of the synthesized strands to be prematurely terminated at the position where dC would normally be added, opposite a template dG.
In the sequencing reaction, DNA synthesis is carried out with what mixture? to extend what primer?
In the sequencing reaction, DNA synthesis is carried out with a mixture of dNTPs and a ddNTP to extend a radiolabeled primer. The products are analyzed by autoradiography to determine the nucleotide sequence.
In the heterozygous BRCA2 mutation of the simulation, which of the following statements is TRUE regarding the Sanger sequencing method detection of the mutation?
In the sequencing reaction, the fragment identifying the mutation and the fragment identifying the wild-type base at that position would be the same length. The Sanger sequencing reaction would have progressed to the same nucleotide for the wild type and for the mutation.
Visualizing topoisomers. BE ABLE TO DESCRIBE THE PICTURE WELL
In this experiment, DNA molecules (plasmids) have an identical number of base pairs but differ in the degree of supercoiling. In lane 1, highly supercoiled DNA migrates as a single band. Lanes 2 and 3 show the effect of treating supercoiled DNA with a type I topoisomerase; the DNA in lane 3 was treated for a longer time than the DNA in lane 2. Each individual band in the bracketed region of lane 3 contains DNA plasmids with the same linking number; Lk changes by 1 from one band to the next.
strand ligation without an activation step, how??
In this scheme, one high-energy bond is replaced by another at each chemical step. The resulting conservation of energy allows strand ligation without an activation step that would otherwise consume ATP.
Poluacrylamide VS agarose
In work on short DNA oligonucleotides (up to a few hundred nucleotides), polyacrylamide is often used instead of agarose as the gel matrix, because it enables researchers to detect small size differences between DNA fragments.
transfection:
Incorporation of exogenous DNA into a eukaryotic cellular genome by any of several methods.
Instead of hydrolyzing a phosphodiester bond
Instead, a nucleophile on the enzyme (usually a Tyr residue, as in the case of E. coli topoisomerase I) attacks the phosphodiester bond displacing the 3′ hydroxyl of one nucleotide and forming a covalent 5′-phosphotyrosyl linkage with the next nucleotide in the DNA strand at the break. Strand passage brings about the topological change. The broken strand is then ligated by a direct attack of the free 3′-hydroxyl group on the phosphotyrosyl linkage.
the head groups of multiple SMC proteins can do what?
Interaction among the head groups of multiple SMC proteins has the potential to produce several different architectures, such as rings, rosettes, and filaments It is not yet clear whether the ringed cohesin tethers around sister chromatids are intra- or intermolecular. The associated proteins may modulate intermolecular interactions, or, for intramolecular rings, they may perform a gatekeeping function in bringing DNA molecules into the ring.
How is Supercoiling not a random process?
It is largely prescribed by the torsional strain imparted to the DNA by decreasing or increasing the linking number relative to that of B-DNA.
what does the Sanger sequencing method require?
It requires a primer for the polymerase; the primer is radiolabelled so the sequences generated can be visualized.
The chemical methods for synthesizing nucleic acids
Knowledge of DNA and RNA chemistry provided the basis for devising methods to synthesize nucleic acids in the laboratory. This technology has paved the way for many biochemical advances that depend on the ability to synthesize oligonucleotides with any chosen sequence. Refinement and automation of these methods have made possible the rapid and accurate synthesis of DNA strands.
larger molecules versus smaller ones??
Larger molecules tend to move more slowly than smaller ones, so this provides a means of separating nucleic acids by size.
Late in the infection process, the viruses produce what?
Late in the infection process, the viruses produce large amounts of two proteins (p10 and polyhedrin)—neither of which is needed for virus production in cultured insect cells, and thus both can be replaced with the gene of a heterologous protein.
Example of uninfected larva and infected larva secondary to the recombinant baculovirus vector
Left: an insect larva infected with a recombinant baculovirus vector expressing a protein that produces a red color. Right: an uninfected larva.
where are many functional RNAs are posttranscriptionally modified at?
Like DNA, many functional RNAs are posttranscriptionally modified at specific nucleotides (Figure 6-34b).
change of lk equals
Lk-Lkinitial
When the linking number changes, some of the resulting strain is usually compensated for by writhe (supercoiling) and some by changes in twist, giving rise to the equation:
Lk= Wr + Tw
FIGURE 9-11: Loops in a eukaryotic chromosome constrained by scaffold proteins. BE ABLE TO EXPLAIN WHAT IS HAPPENING
Loops in a eukaryotic chromosome constrained by scaffold proteins. The chromatin scaffold is attached to the chromosome at intervals, with the DNA between the attachment points defining loops that are topologically constrained.
What indicates that RNA modification has been occurring in biological systems for a long time
Many of the enzymes that catalyze these chemical modifications of RNA have been identified. They are often evolutionarily conserved
function of specialized adaptations of PCR
Many specialized adaptations of PCR have increased the utility of the method.
DNA Methylation tends to inhibit gene expression why?
Methylation tends to inhibit gene expression, because the methylated DNA is not efficiently copied into RNA. In many cancers, gene regulatory regions in DNA become abnormally hypermethylated. This can result in the silencing of genes that would otherwise control cell growth.
Typical flow cell used for a next-generation sequencer.
Millions of DNA fragments can be sequenced simultaneously in each of the eight channels.
How widespread is chemical modification of RNA?
Modified nucleotides in tRNA and rRNA have been recognized for a long time, but we do not know whether other RNAs in cells contain such chemical changes. This is an important question, because modifications could influence the function of RNAs that play various roles in controlling gene expression and therefore might be relevant to understanding disease pathways.
How can biologists figure out the function of these chemical changes in RNA?
Molecular biologists can produce unmodified versions of RNAs in the laboratory and compare their functions with those of the chemically altered counterparts isolated from cells. This approach has only rarely discerned much of an effect of a modified base.
what state do organisms need to stay in? and why?
Most organisms maintain their DNA in a slightly underwound state in order to compact their DNA.
Does Bacteria have multiple sigma factors? If so how are they named? What is the most common?
Named according to their molecular weight the most common is σ70 (Mr 70,000).
figure of relaxed DNA (200) to change in lk +2(202) and change in lk -2 (198)
Negative and positive supercoils. For the relaxed DNA in Figure 9-13a, underwinding or overwinding by two helical turns (ΔLk = ±2) produces negative or positive supercoiling, respectively. Notice that the DNA axis in the two forms twists in opposite directions.
Genomic Sequencing Is Aided by
New Generations of DNA Sequencing Methods
Are covalent bonds broken during denaturation?
No covalent bonds in the nucleic acid are broken during denaturation
Northern blotting can also be used to detect what?
Northern blotting can also be used to detect the levels of a particular type of RNA in different body tissues, providing fascinating insight into how cells regulate the expression of genes.
the sequence obtained for sanger method vs template strand
Note that the sequence obtained is that of the strand complementary to the template strand being analyzed.
Nucleic Acids from Different Species Can what?
Nucleic Acids from Different Species Can Form Hybrids
The Sanger Method Identifies what?
Nucleotide Sequences in Cloned Genes
what can undergo uncatalyzed chemical transformations?
Nucleotides and Nucleic Acids Undergo Uncatalyzed Chemical Transformations
What happens to the nucleic acid once on the nitrocellulose membrane?
Once on the membrane, the nucleic acid can be hybridized with a DNA or RNA probe, labeled so that it can be detected by measuring radioactivity or fluorescence.
Purpose of isolating a gene
One is to examine the DNA itself, determine its sequence, study its structure and/or function, and compare it with other DNA segments. The other possible purpose is to work with the protein or RNA product of the isolated gene.
hints that these RNAs form specific three-dimensional structures important to their function.
One of the key insights from comparative rRNA sequence analysis was the discovery of noncanonical (i.e., non-Watson-Crick) base pairings. Although these had already been observed in tRNA structures, the much larger sizes of rRNA sequences provided vastly more data. For both 16S and 23S rRNAs, much of the sequence could be folded up into base-paired segments. Comparisons between species showed that the base pairings were much more conserved than were the actual nucleotide sequences. This was because a change in the identity of a nucleotide on one side of a base-paired stretch was typically matched by a mutation in its base-pair partner such that base pairing was maintained. Gutell and Woese also noticed that in many cases, such compensatory base changes occurred for base "pairs" not previously thought to form, such as G-U, A-A, and G-A. In this way, long before high-resolution structures of large RNAs became available, it was clear that RNA molecules are much more tolerant of non-Watson-Crick base pairings than is DNA.
RNA polymerase I (Pol I):
One of the three eukaryotic RNA polymerases transcribes genes encoding large rRNA precursors.
RNA polymerase III (Pol III):
One of the three eukaryotic RNA polymerases transcribes genes encoding tRNAs, some snRNAs, 5S ribosomal RNA, and other small functional RNAs.
RNA polymerase II (Pol II):
One of the three eukaryotic RNA polymerases transcribes most of the protein-coding genes.
E. coli has two prominent methylation systems. Describe the first system
One serves as part of a defense mechanism that helps the cell distinguish its DNA from foreign DNA by marking its own DNA with methyl groups and destroys foreign, nonmethylated DNA, a process known as restriction modification.
What would happen if unlabeled dNTPs were not added to the sequencing reaction?
Only the first nucleotide in the sequence would be determined. Because only the ddNTPs are labeled, and because they effectively terminate the reaction, you would be able to determine only the first nucleotide of the sequence; DNA synthesis would be halted at that nucleotide. The unlabeled dNTPs allow the ddNTPs to bind to nucleotides further in the sequence so that you can determine the identity of all the nucleotides in your DNA molecule of interest.
step 3:
Oxidation of the phosphite linkage between the nucleotides completes the cycle
for PCR amplification, how are STR genotypes obtained?
PCR amplification enables investigators to obtain STR genotypes from less than 1 ng of partially degraded DNA, an amount that can be obtained from a single hair follicle, a small fraction of a drop of blood, a small semen sample, or samples that might be months or even many years old.
what does PCR lead to? and how is the amplified segment defined?
PCR leads to specific amplification of DNA in a segment defined by the two designed DNA primers.
PCR primers and flanking DNA
PCR primers are targeted to this flanking DNA and are designed to amplify the DNA across the STR
PCR is useful in identifying suspects in crime scene investigations because:
PCR produces a large amount of DNA for analysis starting from a very small amount. Only a small amount of DNA evidence is typically found at crime scenes, so PCR is useful because the small amount of DNA can be amplified to a quantity that can be worked with, for example, in a sequencing reaction.
what DNA polymerase does PCR use?
PCR uses a heat-stable DNA polymerase, such as the Taq polymerase
Bacterial RNA vs Eukaryotic RNA in regards to enzymes
Pol I, PolI, Pol III are related to bacterial RNA polymerase at the level of both sequence and structure However, the eukaryotic RNA polymerases are larger and contain additional proteins not found in bacteria.
why is probe fluorescence not detectable initially?
Probe fluorescence is not detectable initially due to a fluorescence quencher attached to the same oligonucleotide.
purified closed-circular DNA is rarely relaxed or supercoiled?
Purified closed-circular DNA is rarely relaxed, regardless of its biological origin. Furthermore, all DNA derived from a given cellular source has a characteristic degree of supercoiling
rna duplexes vs DNA duplexes
RNA duplexes are more stable than DNA duplexes.
Part 4 - Prepare for Sequencing
The DNA sample has been purified; your PCR tube should now contain almost nothing but copies of the 16S rDNA. Now, we can prepare the sample for automatic sequencing. DNA sequencing technology is another area of molecular biology that has seen an impressive amount of refinement. The predominant method, illustrated here, is called PCR cycle sequencing. (Learn about cycle sequencing before proceeding.) In this lab, we use a set of 12 primers; six for each strand of the double-stranded DNA. It is theoretically possible to use a single primer in PCR cycle sequencing, but it is not feasible for long sequences. With multiple primers, short, overlapping stretches of DNA are sequenced to obtain the complete sequence. In addition, it is not absolutely necessary to sequence both strands, although sequencing both strands generates redundant data, thereby reducing error. The exact number and location of primers used in a reaction depend on the availability of suitable primers. The primers used here are available from a commercial source and bind to conserved regions of the 16S rDNA gene. They should thus be able to bind to the sequence regardless of the bacterial source. Each green and blue tube contains a "sequencing brew" consisting of buffers, primers (a different one in each tube), DNA polymerases, nucleotides, and fluorescence-tagged terminators in suitable proportions. The PCR product from the previous step is added to each tube and another PCR is run. This time the aim is not to produce identical copies of DNA but many copies of variable length. The animation illustrates what is happening in one tube containing the primer 651R. Each DNA strand binds the primer at one end and will have a fluorescence-tagged terminator at the other end.
What are the DNA sequences flanking STRs unique to? identical?
The DNA sequences flanking STRs are unique to each type of STR and are identical (except for very rare mutations) in all humans.
how does The DNA sequencing method stop its synthesis? and what are the products analyzed on?
The DNA sequencing method uses dideoxynucleotides to terminate synthesis; products are analyzed on a gel.
Partially denatured DNA.
The DNA shown in this electron micrograph was partially denatured, then fixed to block renaturation during sample preparation. The arrows point to some single-stranded bubbles where denaturation has occurred. The regions that denature are reproducible in repeated experiments and are rich in A = T base pairs.
Use of tagged proteins in protein purification: GST (5 steps) more in debt
The GST tag is fused to the C-terminus of the protein by genetic engineering. The tagged protein is expressed in the cell and is present in the crude extract when the cells are lysed. The extract is subjected to chromatography through a matrix with immobilized glutathione. The GST-tagged protein binds to the glutathione, retarding its migration through the column, while the other proteins are washed through rapidly. The interaction between GST and glutathione is tight but noncovalent, allowing the fusion protein to be gently eluted from the column with a solution containing either a higher concentration of salts or free glutathione to compete with the immobilized ligand for GST binding. The tagged protein is subsequently eluted with a solution containing elevated salt concentration or free glutathione.
PCR products and electrophoresis? gel? where?
The PCR products are subjected to electrophoresis on a very thin polyacrylamide gel in a capillary tube.
The PCR products from amplification of up to________ (how many?) STR loci
The PCR products from amplification of up to 16 STR loci can be run on a single capillary acrylamide gel (a "16-plex" analysis).
function of SMC1-SMC3 and SMC2-SMC4 pairs and what are they bound by
The SMC1-SMC3 and SMC2-SMC4 pairs have roles in mitosis All these complexes are bound by regulatory and accessory proteins, including the kleisin family of connector proteins.
The Sanger method uses ddNTPs, which lack ___ __________ _____, to halt ___ _________ at a particular nucleotide
The Sanger method uses ddNTPs, which lack the 3′-hydroxyl group, to halt DNA synthesis at a particular nucleotide.
What does two complementary DNA strands have the ability to do?
The ability of two complementary DNA strands to pair with each other can be used to detect similar DNA sequences in different species or within the genome of a single species.
The ability to hybridize DNA from different species is a valuable for?
The ability to hybridize DNA from different species is a valuable laboratory tool for exploring evolutionary relationships. Different species have proteins and RNAs with similar functions—and often similar structures.
With what can the activity of topoisomers were observed?
The activity of these enzymes can be observed with agarose gel electrophoresis, which separates DNA species according to their topoisomeric form. Topoisomers with Lk values differing by as little as 1 can be separated by this method, so the changes in linking number induced by topoisomerases are readily detected.
The chemical mechanism of RNA synthesis.
The addition of an rNTP to a growing transcript is a Mg2+-dependent reaction that produces a 5′→3′ phosphodiester linkage.
The amelogenin gene:
The amelogenin gene is also used as a marker in the analyses. Present on the human sex chromosomes, this gene has a slightly different length on the X and Y chromosomes. PCR amplification across this gene thus generates different-sized products that can reveal the sex of the DNA donor.
how is the amount of fluorescence in each band is represented??
The amount of fluorescence in each band is represented as a peak in the computer output.
The architecture of RNA polymerases.
The bacterial RNA polymerase core is composed of several subunits that give the enzyme the overall shape of a crab claw. The pincers are formed from the large β and β′ subunits.
what are the bacterial SMC proteins closely related to?
The bacterial SMC proteins are most closely related to condensins. The condensins are critical to chromosome condensation as cells enter mitosis (see Figure 9-25).
How can the absorption of UV light decrease?
The close interaction of stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides and the absorption is further decreased by the pairing of two complementary strands.
Gyrase reaction initiates what?
The complex is then poised to initiate another reaction cycle. The degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoisomerase I, which increases Lk, and DNA gyrase, which decreases Lk.
how do condensins interact with DNA to promote chromosome condensation.
The condensin complexes (SMC2-SMC4 plus associated proteins) first bind to the DNA at the hinge region of the SMC protein, in the interior of what can become an intramolecular SMC ring. ATP hydrolysis then opens the intramolecular ring and brings the DNA inside. Head-to-head association (formed by ATP binding ) creates a structure that traps DNA with a positive superhelical tension. (forming supercoiled loops in the bound DNA. ) Finally, aggregation of the condensins into rosettes forms a condensed chromatid with a defined architecture.
DNA underwinding:
The condition in which a closed-circular DNA has fewer helical turns than would be expected of B-form DNA. Its linking number, Lk, is reduced relative to that of B-form DNA, and the molecule is negatively supercoiled.
Cloning with baculoviruses.
The construction of a typical vector used for protein expression in baculoviruses.
RNA polymerase holoenzyme. Does it exist in many forms or just one?
The core RNA polymerase plus the sigma factor constitutes the RNA polymerase holoenzyme. exists in several forms, depending on the type of σ subunit it contains.
techniques to elongate the sequencing of DNA molecules
The development of two techniques in 1977 (one by Allan Maxam and Walter Gilbert, the other by Frederick Sanger) made possible the sequencing of larger DNA molecules. The techniques depended on the improved understanding of nucleotide chemistry and DNA metabolism and on improved electrophoretic methods for separating DNA strands that differ in size by only one nucleotide
efficiency of each addition step and the implications
The efficiency of each addition step is very high, allowing the routine laboratory synthesis of polymers containing 70 to 80 nucleotides and, in some laboratories, much longer strands.
deamination!
The enzymatic removal of amino groups from biomolecules such as amino acids or nucleotides.
Different between type II topoisomerase and DNA gyrase
The enzyme structure and use of ATP are distinct to this reaction.
The extent of methylation of CpG sequences varies with
The extent of methylation of CpG sequences varies with molecular region in large eukaryotic DNA molecules.
first step in PCR?
The first step in PCR is to design two oligonucleotide primers. Primer sequences are carefully selected so that each primer will anneal to a different strand of the double-stranded DNA template, and will allow polymerase to synthesize DNA across the region of interest.
DNA SUPERCOILING
The folding mechanism must not only pack the DNA but permit access to the information in the DNA in processes such as replication and transcription. DNA is coiled in the form of a double helix, with both strands coiling around an axis. DNA supercoiling is the further coiling of that axis upon itself The coiling of DNA upon itself, generally as a result of bending, underwinding, or overwinding of the DNA helix.
Regulated expression of RecA protein in a bacterial cell. (three steps)
The gene encoding the RecA protein, fused to a bacteriophage T7 promoter, is cloned into an expression vector. Under normal growth conditions (uninduced, left lane), no RecA protein appears when cellular proteins are separated on a polyacrylamide gel and stained with Coomassie Blue for visualization. When the T7 RNA polymerase is induced in the cell (right lane), the recA gene is expressed, and the large amounts of RecA protein produced are readily observed.
more on tag and its function
The gene encoding the target protein is fused to a gene encoding a peptide or protein that binds a simple, stable ligand with high affinity and specificity. The peptide or protein used for this purpose is referred to as a tag. Tag sequences can be added to genes such that the resulting proteins have tags at their N- or C-terminus. Table 7-3 lists some of the peptides or proteins commonly used as tags.
What happens to the gene of interest?
The gene of interest is cloned into a small plasmid between two sites (att) recognized by a site-specific recombinase, then introduced to the baculovirus vector by site-specific recombination. This generates a circular DNA product that is used to infect the cells of an insect larva.
the gene of interest is expressed during what cycle? and where?
The gene of interest is expressed during the infection cycle, downstream of a promoter that normally expresses a baculovirus coat protein at very high levels.
reaction with eukaryotic type II topoisomerases are similar to what? how?
The general mechanism is similar to that of the bacterial DNA gyrase with one intact duplex DNA segment being passed through a transient double-strand break in another segment.
why is there a high concentration of primers?
The high concentration of primers increases the likelihood that they will anneal to each strand of the denatured DNA before the two DNA strands (present at a much lower concentration) can reanneal to each other.
where are amino acid residues located in relation to SMC proteins
The interactions with DNA involve patches of basic amino acid residues near the hinge regions of the SMC proteins.
read length
The length of DNA that can be reliably sequenced in a single cluster by this method—often referred to as the read length, or "read"—is typically 400 to 500 nucleotides, and is constantly increasing.
how can the length of a particular STR be determined?
The length of a particular STR in a given individual can be determined with the aid of the polymerase chain reaction.
what does the length of the PCR product reflect?
The length of the PCR product then reflects the length of the STR in that sample.
Why is DNA a great way to store genetic information?
The long-term storage of information without alteration is so important to a cell, however, that even very slow changes in DNA structure can be physiologically significant.
Why is the temperature for the annealing step in PCR lower than the temperature for the denaturing step?
The lower temperature allows the primers to bind to the template strands, forming hydrogen bonds.
histones
The maintenance of the underwound and condensed state of chromosomes by structural DNA-binding proteins such as histones
after the reaction mixture is heated, what happens? and why?
The mixture is cooled so that the primers can anneal to the DNA.
conserved parts of the polymerase. interior vs exterior
The more conserved parts of the polymerase complex are in the interior, whereas regions that have varied more over the course of evolution are at the exterior of the complex.
reaction catalyzed by eukaryotic type II topoisomerases. steps 1-5
The multisubunit enzyme binds a DNA molecule (step 1). The gated cavities above and below the bound DNA are the N-gate and C-gate, respectively. The second segment of the same DNA is bound at the N-gate (step 2). Both strands of the first DNA are now cleaved (step 3) forming phosphotyrosine intermediates. The second DNA segment passes through the break in the first segment (step 4) and the broken DNA is ligated and the second segment released through the C-gate (step 5). Two ATPs are bound and hydrolyzed during this cycle; it is likely that one is hydrolyzed in the step leading to the complex in step 4.
linking number (Lk)
The number of times one closed circular DNA strand is wound about another; the number of topological links holding the circles together.
sequencing depth or coverage
The number of times that a particular nucleotide in the genome is sequenced, on average, is referred to as the sequencing depth, or sequencing coverage. Although the coverage of particular nucleotides may vary (some will be sequenced 100 times; perhaps a few not at all), this level of coverage ensures that most genomic nucleotides will be sequenced at least 10 times and most sequencing errors will be detected and eliminated.
sequencing depth:
The number of times, on average, that any given sequence among the targeted genomic sequences appears in the sequencing dataset. For example, sequencing to 100-fold coverage indicates that each targeted sequence is represented an average of 100 times in the dataset.
Why is the observed 5% methylation frequency of C residues is lower than would be expected?
The observed 5% methylation frequency of C residues is lower than would be expected based on the random presence of CpG sequences in a genome, and, in fact, CpG dinucleotides occur much less often in vertebrate genomes than predicted by chance. For example, in the human genome, which has a G≡C content of ∼40%, the frequency of CpG would be predicted as 0.2 × 0.2 = 0.04, or 4%, but the frequency is closer to just 1%.
Solid-phase synthesis of nucleic acids.
The oligonucleotide is synthesized in the 3′→5′ direction, starting with a single nucleotide that is covalently attached to a solid support, such as a glass bead (Si). In a repeated series of chemical reactions, nucleotides protected by the dimethoxytrityl (DMT) group from unwanted reactions are sequentially deprotected and reacted to produce a new phosphodiester linkage.
production of double-stranded DNA (dsDNA) copies
The oligonucleotides act as primers for the synthesis of full-length double-stranded DNA (dsDNA) copies of the plasmid that contain the specified sequence change. These plasmid copies are then used to transform cells.
what happens to the other proteins in the extract?
The other proteins in the extract are washed through the column and discarded.
E. coli has two prominent methylation systems. Describe the second system
The other system methylates A residues in the sequence 5′-GATC-3′ to form N6-methyldeoxyadenosine. Methylation in this case is mediated by the Dam (DNA adenine methylation) methylase
denaturation
The partial or complete unfolding of the specific native conformation of a polypeptide chain, protein, or nucleic acid such that the function of the molecule is lost. In the case of nucleic acids, also called melting.
plectonemic and solenoidal supercoiling differences
The plectonemic form is more stable in solution, but the solenoidal form can be stabilized by protein binding and provides a much greater degree of compaction. Instead of the extended right-handed supercoils characteristic of the plectonemic form, solenoidal supercoiling involves tight left-handed turns, much like a garden hose neatly wrapped on a reel.
what then happens to the primed segment?
The primed segment is then replicated selectively by the DNA polymerase, using the pool of dNTPs. The cycle of heating, cooling, and replication is repeated 25 to 30 times over a few hours in an automated process, amplifying the DNA segment between the primers until it can be readily analyzed or cloned.
requirements for primers
The primers you design should each be 20 nucleotides in length, and should anneal to the sequences immediately adjacent to the blue region of interest.
expression of protein in yeast vs bacteria
The principles underlying the expression of a protein in yeast are the same as those in bacteria.
How are probes chosen?
The probe that is selected depends on what is known about the gene under investigation. Sometimes, a gene from another species that has sequence similarity to the gene of interest makes a suitable probe.
DNA genotyping:
The process of defining particular genomic sequences associated with an individual. Also called DNA fingerprinting or DNA profiling.
Part I - SAMPLE PREPARATION
The process of extracting bacterial DNA consists of dissolving the cell wall with a digestive buffer (in the white-capped bottle) available as a commercial kit. The buffer contains proteolytic enzymes that "eat" the cell wall. This step may take several hours. Since we will be using other enzymes in the next step, we need to get rid of the proteolytic enzymes before we can proceed. The enzymes are denatured by heating the sample in a water bath at 100°C. Next, the cellular debris is spun down in the centrifuge and appears as a solid deposit (pellet) at the bottom of the tube. The DNA is contained in the supernatant (the liquid), which is then transferred to the PCR tube.
RecA protein: product of what gene? three functions?
The product of the bacterial recA gene, the RecA protein binds to and forms a filamentous structure on DNA aligns two DNAs of similar sequence hydrolyzes ATP.
DNA topology
The properties of DNA that do not change under continuous deformations.
fusion protein:
The protein product of a gene created by the fusion of two distinct genes or portions of genes. With the use of fusion proteins, almost any protein can be purified by affinity chromatography.
how to alter the rate of expression of a cloned gene?
The rate of expression of the cloned gene is controlled by replacing the gene's own promoter and regulatory sequences with more efficient and convenient versions supplied by the vector.
why is the rate of alteration for purines, pyrimidines, and their nucleotides slow?
The rate of these reactions is generally very slow, but as noted earlier, they are physiologically significant because of the cell's low tolerance for changes in its genetic information.
what then happens to the reaction mixture?
The reaction mixture is heated briefly to denature the DNA, separating the two strands.
What would happen if DNA normally contained uracil?
The recognition of U residues resulting from cytosine deamination would be more difficult and unrepaired uracils would lead to permanent sequence changes as they were paired with adenines during replication (cytosine normally pairs with guanine, so introduction of uracil into DNA effectively changes a C≡G base pair to a U-A base pair).
what do the size of resulting bands secondary to electrophoresis reveal?
The resulting bands are converted into a set of peaks that accurately reveal the size of each PCR fragment and thus the length of the STR in the corresponding allele.
What does the reversible terminator method use to identify nucleotides?
The reversible terminator method of sequencing uses fluorescent tags to identify nucleotides.
role of this ring?
The ring wraps around the sister chromatids, tying them together until separation is required at cell division. The ring may expand and contract in response to ATP hydrolysis.
FIGURE 9-9: Relaxed and supercoiled closed-circular plasmid DNAs. showed on a scanning electron micrograph
The scanning electron micrograph on the far left shows relaxed DNA. Increased supercoiling is shown, from left to right.
Why does DNA contains thymine rather than uracil?
The slow cytosine deamination reaction seems innocuous enough, but it is almost certainly the reason that DNA contains thymine rather than uracil. Establishing thymine as one of the four bases in DNA may well have been a crucial turning point in evolution, making the long-term storage of genetic information possible.
how do you know which one is the primer?
The smallest piece of DNA that has a fluorescent tag attached to it is the primer.
What is the first step of gel electrophoresis?
The starting nucleic acid sample, in solution in a test tube, is applied to a slot at one end of the gel, and a voltage is applied. Because DNA and RNA molecules are negatively charged, they migrate toward the positive end of the gel matrix in the electric field.
Combined DNA Index System (CODIS)
The successful forensic use of STR analysis required standardization, first attempted in the United Kingdom in 1995. The U.S. standard, called the Combined DNA Index System (CODIS), established in 1998, is based on 13 well-studied STR loci, which must be present in any DNA-typing experiment carried out in the United States. By the beginning of 2014, the CODIS database contained nearly 11 million STR genotypes and had assisted more than 220,000 forensic investigations.
Without topoisomerases, cells cannot
The topological state of cellular DNA is intimately connected with its function. Without topoisomerases, cells cannot replicate or package their DNA, or express their genes—and they die.
What can be detected by monitoring the absorption of UV light?
The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring the absorption of UV light.
positive supercoiling:
The twisting of a helical (coiled) molecule on itself to form a left-handed supercoil.
negative supercoiling:
The twisting of a helical (coiled) molecule on itself to form a right-handed supercoil.
Two annealed oligonucleotides serve to..?
The two annealed oligonucleotides serve to prime DNA synthesis in both directions around the plasmid vector, creating two complementary strands that contain the mutation.
how is the sensitivity of PCR useful for crime scenes?
The use of PCR also makes the procedure sensitive enough to be applied to the very small samples often collected at crime scenes.
when is the cloned protein expressed? How does a virus introduce recombinant genome into cells?
The virus uses its natural infection mechanisms to introduce the recombinant genome into cells, where the cloned protein is expressed.
solenoidal supercoiling:
The wrapping of a helical molecule to form a coiled superstructure.
how do the promoter and regulatory sequences direct transcription?
There are many specialized systems for expressing proteins in bacteria. The promoter and regulatory sequences associated with the lactose operon are often fused to the gene of interest to direct transcription.
because cloned eukaryotic genes lack these regulatory processes what needs to happen?
Therefore, appropriate bacterial regulatory sequences for transcription and translation must be inserted at the correct positions, relative to the eukaryotic gene, in the vector DNA. In some cases, cloned genes are so efficiently expressed that their protein product represents 10% or more of the cellular protein.
The simplest examples of supercoiling are provided by small circular DNAs.
These might include plasmids and small, double-stranded viral DNAs.
advantage of proteins: they can be expressed as what two things?
These systems have the advantage that proteins can be expressed either transiently (if the viral DNA is maintained separately from the host cell genome and eventually degraded) or permanently (if the viral DNA is integrated into the host cell genome).
shotgun sequencing.
These technologies are modern manifestations of an approach to genomic sequencing that is sometimes called shotgun sequencing. Many copies of the genomic DNA are sheared to generate each set of fragments. Thus, a particular short segment of the genome may be present in dozens or even hundreds of different sequenced clusters. However, there is no landmark on an individual fragment to indicate where in the genome it came from.
four statements on probes used in hybridization methods
They can be based on DNA sequence information from a sequence database. They can be synthetic DNA sequences based on known amino acid sequences. The probe could be a gene from another species. They are usually radiolabelled or conjugated with a fluorescent tag so they can be visualized.
function of type I enzymes
They function primarily in relieving tension and resolving topological problems in DNA during replication and repair.
how are SNPs important in regards to medicine
This can be critically important in medical and forensic applications, given that two people can share genetic information that differs at only one or a few base pairs, called single-nucleotide polymorphisms (SNPs).
Sanger Method Mechanism
This method makes use of the mechanism of DNA synthesis by DNA polymerases. It requires the enzymatic synthesis of a DNA strand complementary to the strand under analysis, using a radioactively labeled primer and dideoxynucleotides. In the reaction catalyzed by DNA polymerase, the 3′-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond (Figure 7-11a). The identity of the added deoxynucleotide is determined by its complementarity, through base pairing, to a base in the template strand.
Why is the sanger method repeated?
This procedure is repeated separately for each of the four ddNTPs
example of RT-PCR being used?
This reverse transcriptase PCR (RT-PCR) can be used, for example, to detect sequences derived from living cells (which are transcribing their DNA into RNA) as opposed to dead tissues.
how is PCR sensitive?
This technology is highly sensitive: PCR can detect and amplify as little as one DNA molecule in almost any type of sample—including some quite ancient ones.
plectonemic supercoiling can be applied to any structure with
This term can be applied to any structure with strands intertwined in some simple and regular way, and it is a good description of the general structure of supercoiled DNA in solution.
Watson and Crick made extensive use of this image, along with related diffraction data, to develop a model of the three-dimensional structure of DNA that proved to be correct.
This was done at a time before sophisticated computer modeling was possible: Watson and Crick presented their work as a physical model of the double helix constructed on a wire support! Unlike other, competing models of DNA, the Watson-Crick structure had the sugar-phosphate backbone winding around the outside of the helix, with the bases pointing to the interior, where they formed base-pairing interactions between the two strands.
Why is a high temperature needed during the denaturing step in PCR?
To break the hydrogen bonds between the base pairs of the template DNA strands, giving access to the strands for primers to bind
topological linkage/bond
To define linking number, imagine the separation of the two strands of a double-stranded circular DNA. If the two circular strands are interlocked as shown, they are effectively linked by what we define as a topological bond. Even if all hydrogen bonds and base-stacking interactions were removed so that the strands were not in physical contact, the two strands would still be interlocked like the links of a chain link fence.
Step 1 of Gyrase Reaction?
To introduce negative supercoils, a gyrase complex first binds to a DNA segment via the N-gate
Transcription and DNA replication differences
Transcription does not require a primer to begin RNA synthesis Transcription involves defined sections of DNA rather than the entire molecule. Also, just one of the two strands of a DNA segment serves as the template for a given transcription reaction.
advantage of using Transgenic Animals Even large animals
Transgenic Animals Even large animals can be used for the commercial, large-scale production of recombinant proteins. The strategies are different from those discussed thus far and are designed to generate protein in a low-cost, renewable way, such as purification of a protein from the milk of transgenic dairy cattle
Figure of Cross-species DNA hybridization and comparing?
Two DNA samples can be compared by heating a mixture of the DNAs to denature the strands and then cooling the mixture to allow complementary strands to form duplexes. The greater the sequence similarity between the two DNA samples, the more hybrid duplexes will form, with one strand derived from the first species and the other from the second.
catenane:
Two or more circular polymeric molecules interlinked by one or more noncovalent topological links, resembling the links of a chain.
single-nucleotide polymorphisms (SNPs).
Two people can share genetic information that differs at only one or a few base pairs, called single-nucleotide polymorphisms (SNPs).
how DNA strands anneal? within what kind of vector?
Two short, complementary synthetic DNA strands, each with the desired base change, are annealed to opposite strands of the cloned gene within a suitable circular DNA vector. The mismatch of a single base pair in 30 to 40 bp does not prevent annealing.
how are nucleotides added to preexisting strands?
Two synthetic oligonucleotides are prepared, complementary to sequences on opposite strands of the target DNA at positions defining the ends of the segment to be amplified.
Solving topological problems with type II topoisomerases.
Type II topoisomerases resolve knots and catenanes that arise in DNA by passing one duplex through a transient double-strand break in another duplex.
DNA gyrase VS DNA topoisomerase IV
Unlike DNA gyrase, this enzyme does not use ATP and does not introduce negative supercoils.
RNA polymerase catalyzes RNA synthesis in three distinct phases, similar to those of the DNA polymerase reaction.
Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. Initiation occurs as RNA polymerase binds to specific DNA sequences called promoters. Elongation is the process of adding nucleotides to the growing RNA strand. Termination is the release of the product RNA when the polymerase reaches the end of a gene or other transcription unit.
Uracil and the DNA repair system
Uracil is readily recognized as foreign and is removed by a DNA repair system.
What are the functions of noncanonical DNA structures in cells?
We do not yet know whether non-B-DNA functions in specific cellular processes. For example, some evidence suggests that with its left-handed twist, Z-DNA relieves some of the torsional strain that would otherwise build up during DNA transcription. Perhaps for this reason, the potential to form Z-DNA structures correlates with genomic regions of active transcription. But definitive proof of these ideas has been elusive. Whether three-stranded or four-stranded structures are biologically relevant is also a topic that remains ripe for experimentation.
what method does NOT rely on the ability of nucleic acids to hybridize with each other?
Western blotting for the detection of specific proteins that have been separated by gel electrophoresis
What happens when ATP is generated?
When ATP is generated, luciferase catalyzes a reaction with luciferin that results in a tiny flash of light. When many tiny flashes occur in a cluster, the emitted light can be recorded in a captured image.
How to increase UV light absorption (hyperchromic effect)
When DNA is denatured, the base pairs are disrupted and the two strands separate into randomly coiled chains. The resonance of the bases in each strand is no longer constrained, as it is when the bases are part of a double helix. As a result, the UV light absorption of single-stranded DNA is approximately 40% higher than that of double-stranded DNA at the same concentration. This increase in absorption is called the hyperchromic effect.
The origin of negative supercoiling in eukaryotic DNA.
When DNA is wrapped tightly around a DNA-binding protein or protein complex, a solenoidal negative supercoil is fixed in the DNA.
How to name the molecule that has ensured DNA methylation? What element is not identified?
When a chemical group attached to an atom in the purine or pyrimidine ring is altered, the ring position of the substituent is indicated by the number of that atom—for example, 5-methylcytosine, 7-methylguanine, and 5-hydroxymethylcytosine the element to which the substituent is attached (N, C, O) is not identified.
How is the molecule named when a chemical group is altered on an exocyclic atom?
When a chemical group is altered on an exocyclic atom, the type of atom is identified and the ring position to which it is attached is denoted with a superscript. For example, the amino nitrogen attached to C-6 of adenine is N6; the carbonyl oxygen and amino nitrogen at C-6 and C-2 of guanine are O6 and N2, respectively.
relationship between threshold and
When a segment is present in greater amounts in one sample than another, its amplification reaches a defined threshold level earlier. The "No template" line follows the slow increase in background signal observed in a control that does not include added sample DNA.
Why does DNA undergo a physical change?
When a solution is subjected to extremes of pH or to temperatures above 80° its viscosity decreases sharply indicating that the DNA has undergone a physical change.
(steps 4, 5, 6):
When chain synthesis is complete, protecting groups are removed from the bases and phosphates, and the oligonucleotide chain is cleaved from its solid support
accuracy of good STR genotypes
When good STR genotypes are obtained, the chance of misidentification is less than 1 in 1018 (a quintillion).
When does DNA undergo a physical change?
When such a solution is subjected to extremes of pH or to temperatures above 80°C, its viscosity decreases sharply, indicating that the DNA has undergone a physical change. This change is due to denaturation, or melting, of the double-helical DNA and can also occur with RNA.
how to create a a specific DNA sequence change
When suitably located restriction sites are not present, oligonucleotide-directed mutagenesis, coupled to PCR, can create a specific DNA sequence change
What happens when this hinge bends?
When the hinge bends, the α-helical regions form a coiled-coil motif, and the N and C domains come together to form a head structure at one end with a complete ATP-binding site.
how does the fluorescence become detected?
When the reporter oligonucleotide pairs with its complement in a copy of the amplified DNA segment, the fluorophore is separated from the quenching molecule and fluorescence results.
When the resulting recombinant virus is used to infect insect cells or larvae, what happens to the heterologous protein?
When the resulting recombinant virus is used to infect insect cells or larvae, the heterologous protein is often produced at very high levels—up to 25% of the total protein present at the end of the infection cycle.
How do two strands spontaneously rewind? (annealing)
When the temperature or pH is returned to the range in which most organisms live, the unwound segments of the two strands spontaneously rewind to yield the intact duplex.
relaxed DNA
When there is no net coiling of the DNA axis upon itself Any DNA that exists in its most stable and unstrained structure, typically the B form under most cellular conditions.
The polymerase cannot polymerize at this high of a temperature because
While it is true that the polymerase used in PCR can withstand the high temperature needed to melt the template DNA, the polymerase cannot polymerize at this temperature because it cannot bind to the DNA. The polymerase binds to the DNA after the denaturing (melting) step at a cooler, annealing (binding) temperature.
what happens with the correct choice of host cell
With the correct choice of host cell, the proper posttranslational modification of the protein to its active form can be assured.
why is bacteria more convenient than yeast for genetic engineering?
Yeast have tough cell walls that are difficult to breach in order to introduce DNA vectors, so bacteria are more convenient for doing much of the genetic engineering and vector maintenance.
four components of basic PCR
a DNA sample containing the segment to be amplified the pair of synthetic oligonucleotide primers deoxynucleoside triphosphates (dNTPs) DNA polymerase
Some of these vectors incorporate other features such as....
a bacterial ribosome-binding site to enhance translation of the mRNA derived from the gene or a transcription-termination sequence
SMC proteins
a class of enzymes that reversibly form protein loops that link DNA segments, affecting both the condensation/compaction of chromosomes and the cohesion of daughter DNA molecules for periods following replication.
Dam (DNA adenine methylation) methylase
a component of a system that repairs the mismatched base pairs that occasionally form during DNA replication.
Step 2: The 5′-hydroxyl group is temporarily protected by
a dimethoxytrityl (DMT) group
When reading a chromatogram, what is indicative of a mutation in the sequence you are studying?
a double peak of different nucleotides in the same position A DOUBLE peak of DIFFERENT nucleotides in the same position would be indicative of a mutation. The two different peaks at that position would indicate that the DNA sample is heterozygous.
Recall that a cruciform generally contains
a few unpaired bases
agarose
a kelp-derived material that does not disrupt nucleic acid base pairing.
Rosalind Franklin's famous Photograph 51 revealed
a particularly well-resolved x-ray diffraction pattern of a DNA fiber that was interpreted to determine the 3.4 Å distance between base pairs and the 34 Å periodicity of the helix (characteristic of B-form DNA; see Section 6.2) (Figure 1). The darker spots are areas where the film was hit repeatedly with diffracted x-rays from repeating parts of the DNA molecule. At the top and bottom of the photograph, for example, dark patches represent the nucleotide bases of DNA—the patches are dark because the many bases in the DNA fiber are arranged in a regular pattern. The distance between bases in the DNA structure could be determined by measuring the distance between the dark patches on the film and then making a calculation based on how far the DNA sample was from the x-ray film and how it was positioned relative to the direction of the incident x-ray beam.
A column is filled with
a porous matrix consisting of the ligand (glutathione) immobilized on microscopic beads of a stable polymer such as cross-linked agarose.
our understanding of nucleic acid chemistry gives
a powerful array of technologies that have applications in molecular biology, medicine, and forensic science.
our understanding of nucleic acid chemistry gives us
a powerful array of technologies that have applications in molecular biology, medicine, and forensic science.
who created PCR
a process conceived by Kary Mullis in 1983
A probe complementary to the DNA of interest is labeled with
a radioactive or fluorescent functional group.
The gene of interest is cloned into what? linked to what? that in turn directs what?
a special vector, linked to a promoter that directs tissue-specific gene expression. For example, the gene can be placed under the control of regulatory sequences for a mammary gland-specific protein, such as casein lactoglobulin, which is normally secreted in milk in large quantities.
Southern blotting is a method by which
a specific DNA sequence can be identified in a mixture, following size-based fractionation of the DNA sample by agarose gel electrophoresis
DNA sequencing was first automated by
a variation of the Sanger method, in which each of the four dideoxynucleotides used for a reaction was labeled with a differently colored fluorescent tag (Figure 7-12). With this technology, researchers could sequence DNA molecules containing thousands of nucleotides in a few hours, and the entire genomes of hundreds of organisms were sequenced in this way.
how is compaction is maintained
additional specialized DNA-binding proteins, and we turn to these proteins, and their organization and function
Supercoiling occurs in
all chromosomal DNAs in all cells, as well as in viruses that have a double-stranded DNA genome or generate double-stranded DNA as a replication intermediate.
What does the introduction of negative supercoils do?
alone would put additional strain on the DNA molecule
Bacterial topoisomerase II
also known as DNA gyrase can introduce negative supercoils (decrease Lk). This enzyme cleaves both strands of a DNA molecule (thus is a type II topoisomerase) and passes another duplex through the break
After 20 cycles, the DNA segment has been
amplified up to 220, or a millionfold if reaction conditions are ideal.
the sequence of the DNA strand can be read directly from
an autoradiogram of the gel (Figure 7-11c).
Although eukaryotes do not have what?
an enzyme that can introduce negative supercoils into DNA, when a circular DNA is isolated from a eukaryotic cell (e.g., a plasmid from yeast), it is negatively supercoiled. This reflects the generally underwound state of cellular DNA in eukaryotic cells.
nondestructive alterations of DNA
are essential to function such as the strand separation that must precede replication or transcription.
Insect Viruses Baculoviruses
are insect viruses with double-stranded DNA genomes. When they infect their insect larval hosts, they act as parasites, killing the larvae and turning them into factories for virus production.
Ecoli: Topoisomerases I and III
are of type I and they generally relax DNA by introducing transient single-strand breaks to remove negative supercoils (increasing Lk).
how do PCR primers work?
are targeted to sequences on each side of the STR, and the region between them is amplified.
A population of identical plasmid DNAs with the same linking number migrates
as a discrete band during electrophoresis.
Two classes of bacterial topoisomerase inhibitors have been developed
as antibiotics.
Hybridization
base pairing between two strands of nucleic acid from different sources,
Electron micrograph of plectonemically supercoiled plasmid DNA.
be able to recognize this figure
pH and temperature (in regards to experimenting)
be adjusted experimentally to detect nucleic acid sequences with varying degrees of complementarity to the probe.
To be visualized in the laboratory, the probe must
be labeled in some way, usually radioactively or with a fluorophore (a compound carrying a fluorescent group).
Why does the new DNA containing the mutation not have methylated A residues?
because the replication is done in vitro (with no methylating enzymes present).
after three cycles, the target sequences defined by the primers
begins to accumulate after 30 cycles as many as a billion copies of the target sequences are produced from a single starting molecule
This is the opposite of the direction of
biological polynucleotide synthesis by polymerase enzymes, which is 5′→3′.
In addition, chemical synthesis makes it possible to incorporate chemical modifications in the polymer product, such as
biotin groups, extra phosphates, sulfhydryl groups, and methyl groups.
In the linear chromosomes of eukaryotic cells, DNA underwinding is maintained by
bound proteins that constrain the DNA in an elaborate structure called chromatin.
The type I topoisomerase reaction. Bacterial topoisomerase I increases Lk by
breaking one DNA strand, passing the unbroken strand through the break, then resealing the break.
The linking number can be changed by ±1 by
breaking one DNA strand, rotating one of the ends 360° about the unbroken strand, and rejoining the broken ends. This reaction is catalyzed by topoisomerases. Tw and Wr need not be integers. Twist and writhe are geometric rather than topological properties, because they may be changed by deformation of a closed-circular DNA molecule. Tw and Wr may change in a reciprocal manner without altering Lk. That is, Lk can remain unchanged when either Tw or Wr increases by a given amount and the other decreases by that same amount.
deformations do not involve
breaking the DNA
How do specialized enzymes found in all organisms alleviate the stress of replication and transcription??
by introducing or relaxing supercoils.
Transcription in cells and viruses is catalyzed (Prokaryotic)
by specialized enzymes called RNA polymerases
denaturation: RNA duplexes or RNA-DNA hybrid duplexes
can also be denatured.
These 16 STR loci can be run on what type of gel?
can be run on a single capillary acrylamide gel (a "16-plex" analysis).
Deamination of viral nucleic acid
can be used to defend against viral infection.
protease
can remove tag
What can Purines and pyrimidines, and the nucleotides of which they are a part undergo in regards to structure??
can undergo spontaneous alterations in their covalent structure.
PCR can be used quantitatively, by
carefully monitoring the progress of a PCR amplification and determining when a DNA segment has been amplified to a specific threshold level.
underwinding of DNA facilitates
causing supercoiling making strand separation somewhat easier, structural changes in the molecule.
Methylation is generally confined to
certain sequences or regions of a DNA molecule. For example, more than half of all CpG sequences in mammalian genomes are methylated on the C residue.
Bacteria: topoisomerase III class? function?
class: Type I function: has specialized function in DNA repair and replication
eukaryotes: topoisomerase III
class: Type I function: has specialized function in DNA repair and replication
Bacteria: topoisomerase I class? function?
class: Type I function: relaxes negative supercoils
eukaryotes: topoisomerase I
class: Type I function: relaxes negative supercoils, especially during DNA replication
eukaryotes: topoisomerase IiA(alpha)
class: Type II function: Relaxes negative or positive supercoils; functions in chromatin condensation, replication, and transcription
eukaryotes: topoisomerase IiB(beta)
class: Type II function: Relaxes negative or positive supercoils; functions in chromatin condensation, replication, and transcription
Bacteria: topoisomerase IV class? function?
class: Type II function: unlinks replicated chromosomes
how is PCR a useful tool
cloning DNA, forensic medicine, trace ancient human migrations, detecting viral infections before they cause symptoms and for the prenatal diagnosis of a wide array of genetic diseases.
separases
cohesin tethers are removed by enzymes known as separases.
Generally, much of this strain would be accommodated by
coiling the axis on itself, forming a supercoil. 9-10C
How does active DNA underwinding address two biological problems related to DNA?
compaction and information access
continuous deformations
conformational changes due to stretching, twisting, bending, stretching, binding other molecules, thermal motion, or interaction with proteins or other molecules.
general information on Genome projects
continue worldwide, creating rapidly growing online databases containing the complete genome sequences of thousands of organisms.
underwinding contributes to...
contributes to DNA condensation.
methods that rely on the ability of nucleic acids to hybridize with each other?
cross species DNA hybridization for exploring evolutionary relationships colony blot hybridization to identify a specific sequence from a collection of sequences that has been inserted into bacteria Southern blotting for the detection of specific DNA fragments that have been separated by gel electrophoresis Northern blotting for the detection of specific RNA fragments that have been separated by gel electrophoresis
Deamination of cytosine is a common chemical mutation in DNA that can
damage the genetic code if not corrected by the cell.
DNA structure is therefore maintained with
degree of strain that is regulated by the cell and, in turn, induces the observed supercoiling.
twist (Tw)
describes the local twisting or spatial relationship of neighboring base pairs. The net number of helical turns in a DNA molecule.
Modern DNA sequencing methods enable researchers to
determine the sequences of entire mammalian genomes in weeks or even days. Many thousands of genomic DNA sequences are now available in public databases.
Relaxation of unbound positive supercoils by topoisomerases leads to what??
development of a net negative superhelicity in the DNA.
Forensic work focuses on
differences in the lengths of short tandem repeat (STR) sequences.
Different sigma factors are specific for
different promoters
E. coli uses methylation to
distinguish between host and foreign DNA and to facilitate the repair of mismatched base pairs that arise from replication errors.
A key to the discovery of PCR was identifying a DNA polymerase from nature that
does not denature at the high temperature needed to melt the template DNA.
In the Sanger method: results of ddNTP linked to a fluorescent dye
each ddNTP can be linked to a fluorescent (dye) molecule that gives the same color to all the fragments terminating in that nucleotide, a different color for each nucleotide.
When performing automated DNA sequencing, all four sequencing reactions can be analyzed together in the capillary gel because
each ddNTP has a different fluorescent tag.
The underwinding of DNA in vivo has what affect on DNA strand separation?
eases the task of DNA strand separation by enzymes such as DNA and RNA polymerases, and thus facilitates access to the information in the DNA.
The promoter allows
efficient transcription of the inserted gene, and the transcription-termination sequence sometimes improves the amount and stability of the mRNA produced.
To maintain an underwound state, DNA must be
either a closed circle or, if linear, bound to protein.
function of topoisomerases and SMC proteins
enable cells to deal with the complex topological changes occurring as DNA strands separate during replication, repair, and transcription, as well as the extraordinary degree of DNA compaction required in every cell.
SMC Proteins in Eukaryotes
enzymes have integral roles in DNA condensation and chromosome segregation during mitosis, as well as in DNA repair. They perform their tasks by lining up along the DNA and binding to each other, providing a link between distant parts of the chromosome.
heterodimers function in eukaryotes
eukaryotes generally have six SMC proteins that work in defined pairs as heterodimers with different functions (Figure 9-24c).
Alteration of the linking number by
eukaryotic type II topoisomerases.
The supercoiled plasmids tend to be (structure)
extended and narrow rather than compacted, often with multiple branches (Figure 9-17a).
The closer the evolutionary relationship between two species, the more.... (with DNA and hybridization)
extensively their DNAs will hybridize. For example, human DNA hybridizes much more extensively with mouse DNA than with DNA from yeast.
In DNA underwinding, the molecule has
fewer helical turns than would be expected for the B-form structure.
Special strategies are needed to
fill in the inevitable gaps and to deal with repetitive sequences.
how can PCR protocols be made quantitative
for estimating the relative copy numbers of particular sequences in a sample. The approach is called quantitative PCR (qPCR).
The reactions catalyzed by DNA topoisomerases involve the (in regards to structure)
formation of transient covalent DNA-enzyme intermediates, usually in the form of a phosphotyrosyl linkage.
Step 4 of Gyrase Reaction?
forming two 5′-phosphotyrosine intermediates. ATP hydrolysis is coupled to the passage of the second segment of DNA through the cleaved DNA strands, entering at the N-gate and exiting at the C-gate. To complete the reaction
E. coli has at least
four individual topoisomerases, I through IV.
The SMC1-SMC3 pair forms a
functional unit called a cohesin.
The stability of an RNA-DNA hybrid is
generally intermediate between that of double-stranded RNA and DNA.
Topological changes in the linking number are usually accompanied by
geometric changes in both writhe and twist.
The fusion protein is often obtained with
good yield and high purity
gyrase
has an additional activity that uses the energy of ATP to drive key conformational changes that counteract the thermodynamically unfavorable introduction of negative supercoils. Bacterial DNA gyrases are the only topoisomerases known to actively introduce negative supercoils.
The three different eukaryotic SMC proteins form
heterodimers.
Bacterial SMC proteins form a
homodimer.
After transferring the size-fractionated DNA from the gel to a membrane, the probe is
hybridized to the sample on the membrane so that the sequence of interest can be visualized.
Part 3i - Purify PCR Product
i) CONFIRM THAT THE PCR REACTION WORKED The tube should now contain many copies of 16s rDNA, each about 1,500 base pairs (bp) long. At this time, it is prudent to run a gel to confirm that the PCR reaction worked. The gel should contain three lanes: one for the negative control (i.e., water), which should not have a product unless the water was contaminated; another for positive control (PCR product of a known DNA sequence) to make sure that the PCR itself worked; and the last lane for your sample.
What is the main purpose of the bacterial identification virtual lab?
identify disease causing pathogens in patient samples
Applications of southern and northern blotting include
identifying a person on the basis of a single hair left at the scene of a crime or predicting the onset of a disease in an individual decades before symptoms appear.
The DNA sequence is read by .... ?
identifying the color sequences in the bands as they pass the detector. This information is fed directly to a computer, and the sequence is determined.
Inhibitors of topoisomerases have therefore become
important pharmaceutical agents, targeted at infectious organisms and malignant cells.
Hybridization is the basis for
important techniques used to study and isolate specific genes and RNAs.
in cells, plectonemic supercoiling are found in
in smaller DNAs such as plasmids. Larger genomic DNAs are generally compacted in a different way.
DNA replication and transcription similarities
in the direction of synthesis (5′→3′) requirement for a template strand And, like replication, transcription has initiation, elongation, and termination phases.
topoisomerases
influence supercoiling by changing the linking number of chromosomes
In eukaryotes, DNA methylation often
inhibits gene expression.
GyrB
interacts with DNA and ATP and catalyzes ATP binding and hydrolysis. Parts of GyrB form the entry point for DNA, called the N-gate.
Some residues in RNAs are chemically modified by enzymes that ...
introduce methyl or acetyl groups at specific sites or alter a nucleotide base in other ways. These modifications may stabilize RNA structures and can also influence RNA recognition by proteins.
Annealing
involves re-formation of all the base pairs in the double helix.
Writhe (Wr)
is a measure of the coiling of the helical axis, The net number of supercoils in a DNA molecule
An STR locus
is a short DNA sequence, repeated many times in tandem at a specific location in a chromosome; usually, the repeated sequence is 4 bp long.
Glutathione-S-transferase (GST)
is a small enzyme that binds glutathione (a glutamate residue to which a Cys-Gly dipeptide is attached at the carboxyl carbon of the Glu side chain, hence the abbreviation GSH). binds tightly and specifically to glutathione. When the GST gene sequence is fused to a target gene, the fusion protein acquires the capacity to bind glutathione.
function of SMC5-SMC6 pair and what are they bound by
is involved in DNA repair, but its molecular role is not well understood. All these complexes are bound by regulatory and accessory proteins, including the kleisin family of connector proteins.
what is Gel matrix made of?
is not denaturing but instead is made of agarose
The yeast S. cerevisiae
is probably the best understood eukaryotic organism and one of the easiest to grow and manipulate in the laboratory. Like bacteria, this yeast can be grown on inexpensive media.
The DMT group
is removed from the end of the growing polymer chain at the beginning of each cycle to permit extension of the chain by another nucleotide (step 2). Functional groups on the bases and phosphates, including hydroxyl and amine groups, are transiently protected with chemical groups that are readily removed after synthesis is complete.
The elongation step of PCR? temperature relative to annealing and denaturing?
is the step in which the polymerase moves along the strand, adding new nucleotides. The temperature at this step is actually higher than the annealing temperature of PCR.
The melting point (Tm)
is the temperature at which half the DNA in the sample is denatured.
The melting point (Tm) (picture graph)
is the temperature at which half the DNA in the sample is denatured.
origin of negative supercoils in eukaryotic DNA ?
is the tight wrapping of the DNA around a nucleosome in chromatin, which introduces a negative solenoidal supercoil without changing the number of turns in the molecule
gel electrophoresis (another common hybridization method)
is used to separate DNA or RNA molecules by size A technique for separating mixtures of large charged molecules such as proteins or nucleic acids by causing them to move through a gel matrix in an applied electric field.
what does it mean to say the linking number (Lk) is a topological property of double-stranded DNA—that is,
it does not change when the DNA is bent or deformed.
why is Tag polymerase important?
it remains active after every heating step and does not have to be replenished.
Consider, for example, an 84 bp segment of a circular DNA in the relaxed state:
it would contain eight double-helical turns, one for every 10.5 bp 9-10A
The gene of interest is cloned so that ...
its expression is controlled by a virus promoter.
DNA as a respiratory of genetic information depends on
its inherent stability
Cloned genes can be expressed to provide
large amounts of the gene product. Systems have been developed to express genes in bacteria, yeast, insects, mammalian cells, and even entire mammalian organisms.
chromatin and structure
large loops of DNA are constrained at their base, such that each loop is topologically fixed as if it were circular.
Reversible terminator sequencing is placed where? steps?
lies at the heart of the Illumina sequencer. 1. A special sequencing primer is added that is complementary to the oligonucleotides of known sequence that were ligated to the ends of the DNA fragments in each cluster. 2. In addition, fluorescently labeled terminator nucleotides and DNA polymerase are added. 3. The polymerase adds the appropriate nucleotide to the strands in each cluster, each type of nucleotide (A, T, G, or C) carrying a different fluorescent label. 4. These terminator nucleotides have blocking groups attached to the 3′ ends that permit addition of only one nucleotide to each strand. 5. Next, lasers excite all the fluorescent labels, and an image of the entire surface reveals the color (and thus the identity of the base) added to each cluster. 6. The fluorescent label and the blocking groups are then chemically or photolytically removed, in preparation for adding a new nucleotide to each cluster. 7. The sequencing proceeds stepwise.
The reaction of luciferin and ATP with luciferase produces
light flashes when a nucleotide is added to a particular DNA cluster in a particular well.
During mitosis, cohesins
link two sister chromatids immediately after chromosomal replication and keep them together as the chromosomes condense to metaphase
luciferase
luciferase is the enzyme that generates the flash of light produced by fireflies. which in turn generates ATP
denaturation, annealing, and elongation are repeated to
make additional DNA copies
Introduction of negative DNA supercoils by DNA gyrase. (a) The structure of DNA gyrase.
memorize structure
What is an alternative to gel electrophoresis in order to purify PCR products for sequencing?
microfilter column
Supercoiling has been observed in:
most circumstances where double-stranded DNA is present in the cell.
In contrast, all cellular RNA polymerases, from bacteria to humans, are composed of
multiple polypeptides that fold together to create the functional enzyme.
Bacterial DNA gyrases introduce
negative supercoils.
Negative linking numbers are, for all practical purposes
not encountered in DNA.
Solid-phase synthesis of DNA and RNA
occurs in the 3′→5′ direction, using chemically protected nucleotides that are selectively deprotected and coupled to the growing polynucleotide chain in successive cycles.
Double-stranded DNA absorbs less UV light than single-stranded DNA because:
of the hypochromic effect that results when hydrogen bonding and base stacking in double-stranded DNA limits resonance in the aromatic rings of the bases.
Native DNA undergoes reversible unwinding and separation of the strands (melting) under what conditions?
on heating or at extremes of pH.
Base-pairing stability is sensitive to
pH and temperature
What chemical reaction is prevented by the incorporation of ddNTPs into the sequencing reaction?
phosphodiester bond formation The ddNTPs differ from normal DNA nucleotides in that they lack the 3'-hydroxyl group on the pentose ring. Without this hydroxyl group, the next phosphodiester bond in the reaction cannot form.
DNA methylation may affect gene transcription by
physically blocking the binding of proteins that facilitate transcription. Other proteins, however, can specifically bind to methylated DNA and recruit additional proteins that help form compact, inactive regions of chromosomal DNA.
For RNAs, chemical modifications can
play significant roles in ensuring correct structure and function.
In a constrained DNA molecule, what needs to happen??
positive supercoils must develop elsewhere to compensate for the resulting strain.
components of a polymerase chain reaction (PCR)?
primers complementary to each end of the sequence to be amplified heat stable Taq polymerase DNA ligase to connect the fragments together
Plectonemic supercoiling does not
produce sufficient compaction to package genomic DNA in the cell.
Solenoidal supercoiling, an alternative form that
produces a greater degree of compaction, is characterized by tight left-handed turns that are stabilized by wrapping the DNA around proteins; this occurs in eukaryotic and bacterial chromosomes.
SMC Proteins
promote chromosome condensation by creating physical contact between segments of DNA that may otherwise be quite distant from each other in the chromosome, or even on different chromosomes. This class of protein is found in all organisms. A family of ATPases that modulate the structure and organization of chromosomes.
cellular function of cytosine deamination
provides innate cellular defense against viral infection
Renaturation of a DNA or RNA molecule
rapid, one-step process, as long as a double-helical segment of at least a dozen residues still unites the two strands.
RFU =
relative fluorescence units, measured against a standard supplied with the kit.
topoisomerase play an especially important role in the complex changes in DNA topology during
replication and DNA packaging.
underwinding allows access to DNA during
replication and transcription
FOR EXAMPLE, what is the function of restriction endonuclease cleavage sites
restriction endonuclease cleavage sites can be included to facilitate the subsequent cloning of the amplified DNA
methods used to amplify RNA sequences
reverse transcriptase-PCR using DNA primers
Plectonemic supercoiling includes
right-handed branches and is the most common type of supercoiling in isolated DNA.
In the laboratory, the supercoils are? example?
right-handed in a negatively supercoiled DNA molecule, such as a supercoiled plasmid isolated from a bacterium.
In principle, the strain induced by this degree of net DNA underwinding could also be accommodated by
separating the two DNA strands over a distance of about 10 bp (Figure 9-10d).
The operator (O) is a
sequence bound by a protein called a repressor, which normally blocks gene expression from the adjacent gene.
The ribosome-binding site provides
sequence signals for the efficient translation of the mRNA derived from the gene.
ribosome-binding sites
sequences that allow translation of the mRNA to protein
promoters
sequences that instruct RNA polymerase where to bind to initiate mRNA synthesis
The efficiency of genomic sequence generation comes from what?
sequencing all of these millions of clusters at the same time, with the data from each cluster captured and stored in a computer.
function of the oligonucleotides
serve as replication primers that can be extended by a DNA polymerase. The 3′ ends of the hybridized primers are oriented toward each other and positioned to prime DNA synthesis across the targeted DNA segment
The loci most often used in STR genotyping are .....
short—4 to 50 repeats (16 to 200 bp for tetranucleotide repeats)—and have multiple length variants in the human population. More than 20,000 tetranucleotide STR loci have been characterized in the human genome. And more than a million STRs of all types may be present in the human genome, accounting for about 3% of all human DNA.
DNA and RNA polymers of any sequence can be synthesized with
simple, automated procedures involving chemical and enzymatic methods.
What would happen to some of the strain in this 84 bp segment?
simply become dispersed in the untwisted structure of the larger DNA molecule.
The resulting colored DNA fragments are separated by
size in an electrophoretic gel in a capillary tube (a refinement of gel electrophoresis that allows for faster separations).
Gel electrophoresis is used to
size-fractionate a DNA (Southern blotting) or RNA (Northern blotting) mixture. The samples are then transferred to (i.e., are blotted onto) a nitrocellulose membrane, where they are detected using short radiolabeled oligonucleotide probes that base-pair to the samples on the membrane.
forms that can be adopted by an underwound DNA in the cell?
solenoidal and plectonemic supercoiling, can be adopted by an underwound DNA in the cell
Problems with proteins when expressed in bacteria
some heterologous proteins do not fold correctly, and many do not undergo the postsynthetic modifications (covalent modification, proteolytic cleavage) necessary for their activity. A variety of gene sequence features also can make a particular gene difficult to express in bacteria. For these and many other reasons, some eukareryotic proteins are inactive when purified from bacteria, or they cannot be expressed at all
What are these functional groups useful (biotin groups, extra phosphates, sulfhydryl groups, and methyl groups)????
specific labeling of a DNA strand or stabilization of an RNA oligonucleotide against enzymatic degradation in cells.
DNA underwinding facilitates
strand separation by enzymes of DNA metabolism.
Supercoiling and Structural Strain
supercoiled DNA (sometimes called superhelical DNA) is generally a manifestation of structural strain. The DNA can be overwound to generate supercoils. underwinding of the DNA also imparts strain and results in supercoils. .
Most cellular DNAs are
supercoiled.
measurable properties of supercoiling
supercoiling has provided many insights into DNA structure and function.
In isolated closed-circular DNA, strain introduced by underwinding is generally accommodated by what? and Why?
supercoiling rather than strand separation, because coiling the axis of the DNA usually requires less energy than breaking the hydrogen bonds that stabilize paired bases.
In addition to the tendency of methylation of C residues to inhibit gene expression, methylation does what other function?
suppresses the migration of segments of DNA called transposons.
function of DNA polymerases
synthesize DNA strands on a pre-existing DNA template using free deoxyribonucleotides. Further, DNA polymerases do not synthesize DNA de novo, but instead must add nucleotides to preexisting strands, referred to as primers
effect of condensing binding to DNA
that is, condensin binding causes the DNA to become overwound, in contrast to the underwinding induced by the binding of nucleosomes.
The use of different radiolabeled rNTPs revealed
that only ATP and UTP were needed for complete synthesis of RNA, and not GTP or CTP
ahead the enzyme (direction of transcription)
the DNA becomes overwound ahead of the enzyme (upstream; positive supercoils)
Steps following electrophoresis.. what happens and why??
the DNA fragments are transferred to a nitrocellulose membrane so that their positions in the gel relative to each other are preserved on the membrane.
The underwound state can be maintained only if
the DNA is a closed circle or, if linear, is bound and stabilized by proteins so that the strands are not free to rotate about each other.
Most cloned eukaryotic genes lack
the DNA sequence elements required for their controlled expression in bacterial cells—promoters, ribosome-binding sites, and additional regulatory sequences At these concentrations, some foreign proteins can kill the host cell (usually E. coli), so the cloned gene expression must be limited to the few hours before the planned harvesting of the cells.
The Gyrase Reaction is now over but what still happens
the DNA strands are ligated by attack of the free 3′-hydroxyl groups on the phosphotyrosine intermediates.
what happens if the sequence of interest is present at higher levels than other sequences in the sample
the PCR signal will reach a predetermined threshold faster.
Gene Sequences Can Be Amplified with
the Polymerase Chain Reaction
Topological properties of DNA can be changed only by
the breakage and rejoining (religation) of the backbone of one or both DNA strands.
when is the cloned gene transcribed
the cloned gene will be transcribed when lactose is added to the growth medium.
Assembling the sequences of these millions of fragments into a genomic sequence requires what?? How do overlaps help?
the computerized alignment of overlapping fragment sequences The overlaps allow the computer to trace the sequence through a chromosome, from one fragment to another. This permits the assembly of long contiguous sequences called contigs.
The denaturation of a double-helical RNA often requires
the denaturation of a double-helical RNA often requires higher temperatures, by 20°C or more, than those required to denature a DNA molecule with a comparable sequence.
The cleft between the two pincers of the claw contains ...
the enzyme active site and binds two Mg2+ ions that facilitate RNA polymerization.
comparing ribosomal RNA sequences would provide valuable information about
the evolutionary relationships among different organisms.
DNA Helices Have Unique Geometries That Depend on Their Sequence
the first single crystal of a DNA molecule was an important landmark because for the first time it became possible to determine the exact helical parameters of a defined DNA sequence. Once the methodology was available to synthesize DNA oligonucleotides on solid supports, short DNA molecules of specific length and sequence could be produced in milligram quantities. This material could be purified, and it crystallized readily when concentrated slowly in the presence of suitable buffers. DNA fibers can readily form from a mixture of DNAs of different lengths and sequences, and the structures obtained by analyzing the fiber diffraction patterns produce an "averaged" structure of all the molecules in the fiber. In contrast, single crystals, by definition, are formed by arrays of identical molecules. Richard Dickerson and his colleagues used a self-complementary dodecamer sequence, CGCGAATTCGCG, to solve the first single-crystal structure of DNA.
Underwinding of a right-handed DNA helix also facilitates
the formation of short stretches of left-handed Z-DNA in regions where the base sequence is consistent with the Z form
fragments near the bottom of the gel represent? and how is the sequence read?
the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 5′ end) and the sequence is read (in the 5′→3′ direction) from bottom to top.
DNA (or RNA) synthesis is carried out with what???
the growing strand attached to a solid support
The amount of PCR product present is determined by measuring
the level (fluorescence) of a fluorescent probe attached to a reporter oligonucleotide complementary to the DNA segment that is being amplified.
Underwinding is quantified by
the linking number (Lk), a topological parameter that describes the number of times two DNA strands are intertwined.
Lk equation
the linking number is equal to the number of base pairs divided by the number of base pairs per turn
The amplified DNA can then be cloned by
the methods described earlier or can be used in a variety of analytical procedures.
Because the Lk remains unchanged, what needs to happen to the negative solenoidal supercoil?
the negative solenoidal supercoil has to be compensated for by a positive supercoil elsewhere in the DNA The type II topoisomerases relax the unbound positive supercoils that arise in this way. The bound and stabilized negative supercoils are left behind, conferring a net negative superhelicity on the DNA.
The change in linking number has no effect on
the number of base pairs or the number of atoms in the circular DNA molecule.
Sigma factors play an important role in
the recognition of different types of bacterial genes
Reverse transcriptase PCR and quantitative PCR can be combined to determine what?
the relative transcription levels of genes in a cell under different environmental conditions OR to study the regulation of transcription of one or more genes.
The selectable marker allows
the selection of cells containing the recombinant DNA.
Underwinding is measured in terms of σ,
the superhelical density; σ = ΔLk/Lk0.
Underwinding decreases
the total number of helical turns in the DNA relative to the relaxed, B form.
Topoisomerases catalyze
the underwinding and relaxation of DNA. On a molecular level, topoisomerases catalyze changes in the linking number
Electron micrographs of different bacterial SMC dimers show what?
the variety of shapes these dimers can take. Electron microscopy reveals the flexible V shape of these proteins part D
Solenoidal negative supercoiling consists of
tight left-handed turns about an imaginary tubelike structure.
Cloned Genes Can Be expressed to do what?
to Amplify Protein Production
Notably, these classical methods of Southern and Northern blotting are still used
to answer specific experimental questions, despite the development of high-throughput strategies based on DNA sequencing technology.
adjusting pH and temp is sensitive enough
to reveal sequences that differ by a single base pair.
topoisomerase in bacteria
topoisomerase I, II (DNA Gyrase), III, IV
topoisomerase in eukaryotes
topoisomerase I, IiA(alpha), IiB(beta), III
The type I enzymes are called
topoisomerases I and III.
The type II enzymes are
topoisomerases IIα and IIβ
What happens if the STR sequences have different lengths on the two chromosomes of an individual's chromosome pair
two PCR products of different lengths result.
In E. coli, for example, the RNA polymerase core is a large, complex enzyme with five polypeptide subunits:
two copies of the α subunit and one copy each of the β, β′, and ω subunits: α2ββ′ω (Mr 390,000). exclusive of the σ subunit
Gyrase is composed of what?
two types of subunits, GyrA and GyrB, functioning as a GyrA2GyrB2 heterotetramer.
Eukaryotic cells also have
type I and type II topoisomerases.
Next to the histones that make up the nucleosomes what is the most abundant proteins?
type II topoisomerases are the most abundant proteins in chromatin.
Solenoidal supercoiling is the primary mechanism by which
underwinding contributes to genomic DNA compaction.
In almost every instance in nature, the strain is a result of
underwinding of the DNA double helix in the closed circle.
Behind the enzyme
underwound behind it (downstream; negative supercoils). Red arrows indicate the direction of winding.
Gel electrophoresis used in what techniques?
used in the Southern and Northern blotting techniques.
This structure, known as the Dickerson dodecamer
ushered in an era of high-resolution structural determinations of DNA and, eventually, the crystallographically determined structures of specific DNAs bound to protein partners. The study of individual DNA sequences also led to extensive studies of DNA-small molecule interactions and to research on the effects of DNA mutations on helical geometry. This work guided the development of certain anticancer drugs, such as cisplatin, that bind and distort DNA and thereby disrupt its replication in rapidly growing cells.
A variety of engineered mammalian viruses are available as
vectors, including human adenoviruses and retroviruses.
The PCR Master Mix solution contains the following:
water a buffer to keep the mixture at the correct pH for the PCR reaction large quantities of the four nucleotides adenine, cytosine, guanine, and thymine large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to initiate the replication process a heat-stable DNA polymerase that extends the copy DNA strand.
The Sanger method makes use of ddNTPs,
which lack a 3'-hydroxyl group, to halt DNA synthesis at a particular nucleotide.
example, σ = −0.01 means
which means that 1% (2 of 200) of the helical turns present in the DNA (when it is relaxed, in its B form) have been removed.
The most convenient way to introduce cloned genes into a mammalian cell is? why?
with viruses. In this way, a molecular biologist can take advantage of the natural capacity of a virus to insert its DNA or RNA into a cell, and sometimes into the cellular chromosome.
In a closed-circular DNA molecule, however, the number of helical turns cannot be changed without doing what?
without at least transiently breaking one of the DNA strands. The number of helical turns in a DNA molecule therefore provides a precise description of supercoiling.
Step 2 of Gyrase Reaction?
wraps the DNA around itself
We can break down the linking number into two structural components
writhe (Wr) and twist (Tw)
Strain from underwinding of the DNA can manifest as
writhe or twist.
The molecular structures of bacterial and yeast RNA polymerases have been determined by
x-ray crystallography
For cellular DNAs, σ is typically
−0.05 to −0.07, which means that about 5% to 7% of the helical turns in the DNA have been removed.
