Biochem SG 8 and 9

Describe how PCR works. RTPCR and qPCR work.

-Basic PCR requires four components: 1) a DNA sample containing the segment to be amplified, 2) two synthetic oligonucleotide primers, 3) dNTPs, and 4) DNA polymerase. This reaction mixture is heated briefly to denature the DNA (doesn't denature the DNA polymerase TaqI), separating the two strands. The mixture is cooled so that the primers can anneal to the DNA. The high concentration of primers increase the likelihood that they will anneal to each strand of the denatured DNA before the two DNA strands can anneal to each other. The primed segment is then elongated selectively by the DNA polymerase, using the pool of dNTPs. This entire cycle is repeated 25-30 times over a few hours. DNA amplified by PCR can be cloned because the primers can include noncomplementary ends that have a site for cleavage by a restriction endonuclease. Although these parts of the primers do not anneal to the target DNA, the PCR process incorporates them into the DNA that is amplified. Cleavage of the amplified fragments at these sites creates sticky ends, used in ligation of the amplified DNA to a cloning vector. -In RT-PCR (Reverse Transcription-Polymerase Chain Reaction), first, reverse transcriptase and primer will anneal to and transcribe mRNA to a complimentary strand of DNA. This DNA strand is then replicated with primers and Taq Polymerase, and the standard PCR protocol is followed. The PCR products (the DNA strands) are then separated with agarose gel electrophoresis and if a band shows up for the desired molecular weight, then the mRNA was in fact present in the sample, and the associated gene was being expressed. -Quantitative PCR is carried out much in the same way as regular PCR (but the amplified DNA is detected as the reaction progresses in "real time."): heated to allow denaturation, cooling to allow binding of the primers with DNA template, and another slight heating to facilitate the polymerization. However, in qPCR, a fluorescent probe is attached to a reporter oligonucleotide complementary to the DNA segment that is being amplified. When the reporter oligonucleotide pairs with its compliment in a copy of the amplified DNA segment, fluorescence results. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified.

Explain Chargoff's rule, and relate it to the structure of DNA. How is this the same as Watson-Crick Base pairing?

-Chargoff's rule: -found that 4 nucleotide bases of DNA occur in different ratios in the DNA of different organisms an that the amounts of certain bases are closely related. 1. The base composition of DNA generally varies from one species to another 2. DNA specimens isolated from different tissues of the same species have the same base composition 3. The base composition of DNA in a given species does not change with an organisms age, nutritional state, or changing environment 4. In all cellular DNAs, regardless of species, the number of adenosine residues is equal to the number of thymidine residues (A=T or U) and G=C. From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues (A+G=T+C) -Watson and Crick relied on Chargoff's rule to set about deducing DNA's 3D structure. They found that the H-bonded base pairs, G with C and A with T, are those that fit best within the structure, providing a rationale for Chargoff's rule that in any DNA, G=C and A=T. The two antiparallel polynucleotide chains of double-helical DNA are complementary: wherever A occurs in one chain, T is found in the other (same for G and C). Also, three H-bonds can form between G and C but only two can form between A and T. Hydrophobic stacking interactions.

How do the ribose sugars in DNA and RNA differ?

-DNA has a deoxyribose sugar and RNA has a ribose sugar -DNA is a modified sugar, lacking one O atom (this difference allows enzymes to distinguish them) -RNA has a hydroxide group on the 2' C -DNA lasts longer in water because backbone is more stable because in RNA there are two lone pairs on the O that can attack the P and lead to cleavage of the backbone. -Nucleic acids have two kinds of pentoses. The recurring deoxyribonucleotide units of DNA contain 2'- deoxy-D-ribose, and the ribonucleotide units of RNA contain D-ribose.

From 'melting' of DNA what enzyme did we realize had to exist? What 'chaperone' like protein needed also to exist? What makes RNA polymerase unique in this regard?

-DNA helicase is an enzyme that must exist. It catalyzes and is responsible for the opening the DNA strands (it forms single-stranded DNA) to allow for DNA replication and transcription. DNAa recognizes ORI and opens up the DNA molecule. DNA polymerase is a "chaperone" like protein that must exist as well. It recreates/elongates the new strand of DNA from a single strand or primer. RNA polymerase is unique because it has helicase activity and does not require another enzyme like DNA polymerase.

What is the Tm of DNA due too, which base pairs is it dependent upon, and why?

-Each species of DNA has a characteristic denaturation temperature, or melting point (tm) (where half of the DNA strands are in the random coil or single-stranded state). The Tm is affected by a number of factors: concentration of ions in the solution (most notably Mg+ and K+), DNA sequence, and the length of DNA. The higher its content of G-C base pairs, the higher the melting point of the DNA. This is because G-C base pairs, with three hydrogen bonds, require more heat energy to dissociate than A-T base pairs.

Explain Introns and Exons.

-Exons are nucleotide sequences encoded by a gene that remains present in the final mature RNA after RNA splicing, when introns are removed and exons are covalently joined to one another as the product RNA. Exons contain part of the open reading frame (ORF) that codes for a specific portion of the complete protein. -Introns are nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA product of a gene is being generated. Some introns themselves encode specific proteins or can be further processed after splicing to generate noncoding RNA molecules. Introns reduce the chance of mutation among the eons. Exons code for proteins, whereas introns do not directly code for proteins.

There are posted on WORDPRESS papers about miRNA and iRNA. What are there functions? Structure?

-MicroRNA (miRNA, ~22 base pairs in length) and short interfering RNA (siRNA.~21 base pairs in length) are forms of non-coding RNA involved in RNA interference (RNAi), a major component of the post-transcriptional regulation of genes -miRNA, or microRNA, is a small (about 21 nucleotides long), non-coding RNA molecule found in plants and animals, which functions in transcriptional and post-transcriptional regulation of gene expression. In mammals, miRNAs are predicted to control the activity of ~50% of all protein-coding genes. They are encoded by eukaryotic nuclear DNA and function via base pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation. The gene that codes for a miRNA first produces a ~70 nucleotide transcript. This "pre-miRNA" transcript has the capacity to form a stem-loop structure. This pre-miRNA is then processed into 21-22 nucleotide long miRNA by an enzyme called Dicer. There is a seed region of about 6-8 nucleotides long at the 5' end of the miRNA that is involved in target specificity. MiRNAs are not entirely complementary to their target mRNA transcripts; therefore, they are able to knockdown the regulation of multiple mRNA transcripts. MiRNAs are bound by an argonaute protein to form the RISC complex and bind to mRNA to inhibit translation. Accumulation of multiple miRNAs on to one transcript is a signal for the cell to relocate the target mRNA to P-bodies which are sites of mass mRNA degradation. -SiRNAs are entirely complementary to their target gene; therefore they are specific to a certain transcript. The siRNA-RISC directly cleaves target mRNA transcripts resulting in their direct degradation.siRNAs have a well-defined structure: a short (usually 20 to 24-bp) double-stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides.

What is the origin of replication (ori)?

-ORI is a particular sequence in a genome at which replication is initiated. This sequence is required to propagate the plasmid and maintain it at a level of 10 to 20 copies per cell. ORIs are found in prokaryotes and eukaryotes, or that of DNA or dsRNA in viruses. The specific structure of the origin of replication varies somewhat from species to species, but all share some common characteristics such as high AT content. Two types: 1) narrow or broad host range and 2) high- or low- copy number. The origin of replication binds the pre-replication complex, a protein complex that recognizes, unwinds, and begins to copy DNA.

What is an otholog, a paralog?

-Ortholog: Genes from different species but possess a clear sequence and functional relationship to another (for example: a newly discovered gene is related by seq homologies to a gene previously studied in another or same specie and its function can be entirely or partly defined by that relationship). The identity is easiest to make when comparing genomes from relatively closely related species, such as a mouse and a human. Normally, orthologs retain the same function in the course of evolution. -Paralog: Genes of different species that are related by duplication within a genome. They derive from different ancestral genes. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one. Paralogous genes are presumed to have been derived by gene duplication fol- lowed by gradual changes in the sequences of both copies. Typically, paralogous proteins are similar not only in sequence but also in three-dimensional structure, although they commonly have acquired different functions during their evolution.

How are miRNA and siRNA produced in a cell?

-RNA polymerase II transcribes a section of non-coding DNA. This long precursor RNA molecule is then processed in the nucleus by Drosha to produce a primary miRNA (pri-miRNA). Pri-miRNA can contain the sequence for multiple miRNAs. Exportin5 allows the pri-miRNA to pass through the nuclear pore into the cytoplasm. Once in the cytoplasm, a protein complex made up of an RNA binding protein TRBP and an RNA-specific RNase called Dicer process the pri-miRNA into an miRNA. One of the two strands is chosen to be bound by an Argonaute protein to produce the RNA Induced Silencing (RISC) Complex. This complex is what binds the target mRNA and inhibits translation. -The Dicer enzyme catalyzes production of siRNAs from long dsRNAs and small hairpin RNAs into 20-21 bp fragments that are then loaded on the complementary RNA target by the RISC complex. This induces degradation of the target mRNA. The dsRNA is processed in the cytoplasm by Dicer to form siRNAs. One strand of the SiRNA associates with the same RISC complex; however, the siRNA-RISC complex is able to directly cleave a target mRNA transcript. siRNAs can also be introduced by transfection.

What are hybrid heteroduplexes? What can you do with this phenomena?

-A hybrid heteroduplex is a double stranded molecule of nucleic acids (DNA or RNA) that has been combined together from single complementary strands derived from different sources, or organism. (example was segments of a mouse DNA strand form base-pairing regions with segments of a human DNA strand). The isolation and identification of specific genes and RNAs rely on these hybridization techniques: a specific DNA sequence or gene can be detected in the presence of many other sequences if one already has an appropriate complementary DNA strand to hybridize with it. Applications of this technology also make possible the identification of an individual on the basis of a single hair left at the scene of a crime or the prediction of the onset of a disease decades before symptoms appear. -This reflects a common evolutionary heritage; different organisms generally have some proteins and RNAa with similar functions, and, often, similar structures. In many cases, the DNAs encoding these proteins and RNAs have similar sequences. The closer the evolutionary relationship between two species, the more extensively their DNAs will hybridize.

Why are Selectable Marker necessary? Antibiotic resistance genes? Β-glactosidase?

-A selectable marker is a reporter gene introduced into a cell along with a gene insert (the desired gene of study). They are necessary because the experimenter can tell if the right gene is in the cell because the marker can be seen or detected. Selectable markers are often antibiotic resistance genes; bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic. The antibiotic knocks out cells that do not have the resistant marker. Those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material -Antibiotic resistance genes: allows identification of cells that contain the intact plasmid or a recombinant version of the plasmid. -β-galactosidase is an exoglycosidase which cleaves the β-glycosidic bond formed between a galactose and its organic moiety. X-gal produces a characteristic blue dye when cleaved by β-galactosidase, thereby providing an easy means of distinguishing the presence or absence of active Beta-galactosidase/cloned product in a plasmid with the naked eye. It is used as a reporter marker to monitor gene expression.

Explain the structure of the following co-enzymes (co-substrates), CoA, FAD, NAD, and FMN.

-All unrelated structurally except for the presence of adenosine. Adenosine does not participate directly in the primary function, but removal of adenosine generally results in a drastic reduction of cofactor activities. Once ATP became the universal source of chemical energy, systems developed to synthesize ATP in greater abundance than other nucleotides and therefore becomes a logical choice for incorporation into a variety of structures -CoA- Coenzyme A functions in acyl group transfer reactions; the acyl group (such as the acetyl or acetoacetyl group) is attached to the CoA through a thioester linkage to the β-mercaptoethylamine moiety. - FAD, the active form of vitamin B2(riboflavin), in electron transfers. Another coenzyme incorporating adenosine is 5'-deoxyadenosylcobalamin, the active form of vitamin B12, which participates in intramolecular group transfers between adjacent carbons. -NAD: functions in hydride transfers -FMN: biomolecule produced from riboflavin vitamin B2 by the enzyme riboflavin kinase.

Explain mutations? How does that help in our understanding the role of specific amino acids in a protein?

-Alterations in DNA structure that produce permanent changes in the genetic information encoded therein are called mutations. Mutations result from unrepaired damage to DNA or RNA genomes typically caused by radiation, chemical mutagens, oxidative damage, errors in the process of replication, or from insertion or deletion of segments of DNA. Mutations may affect or have no effect on the product of the gene and may be beneficial or harmful to the organism. It may or may not change the phenotype. Induced mutation (site directed mutagenesis) can help scientists deduce the roles of specific amino acids by altering certain genes and observing the effect on the organism -Changes in the DNA sequences change the mRNA sequence and thus the protein sequence, Changes in the protein sequence cause changes in functionality. Without proper functionality, we can examine which functions are protein may involved with.

What is a BLAST of a particular gene? Why is the e value important? (also from lecture).

-Basic Local Alignment Search Tool: is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences A BLAST search enables a researcher to compare a query sequence with a library or database of sequences, and identify library sequences that resemble the query sequences above a certain threshold. (ex: discovery of unknown gene in a mouse, a scientist will perform a BLAST search of the human genome to see if humans carry a similar gene; BLAST will identify sequences in the human genome that resemble the mouse gene based on similarity of sequence). - The Expectation value or Expect value represents the number of different alignments with scores equivalent to or better than S that is expected to occur in a database search by chance. E value is a statistical parameter which accounts for the significance of an alignment. Lesser the e value, least the probability that the alignment is a result of mere random chance. The number of hits one can "expect" to see just by chance when searching a database of a particular size.

Looking at your answer to question 12, explain site-directed mutagenesis.

-Site-directed mutagenesis is a method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products in order to decipher which amino acids account for which activities in a protein. It is used for investigating the structure and biological activity of specific DNA, RNA, and protein molecules, and for protein engineering. -cloning techniques can be used to produce protein products subtly altered from their native forms. Specific amino acids may be replaced individually by site-directed mutagenesis. This changes the amino acid sequences of a protein by altering the DNA sequence of the cloned gene. If appropriate restriction sites flank the sequence to be altered, researchers can simply remove a DNA segment and replace it with a synthetic one that is identical to the original except for the desired change (oligonucleotide-directed mutagenesis when restriction enzymes are not present). -How: A short DNA primer contains the desired mutation and is complementary to the template DNA around the mutation site (not compl. to mutation!). The mutation may be a single base change, multiple base changes, a deletion or an insertion. DNA polymerase extends the single-stranded DNA primer and copies the rest of the gene along with the mutation. Amplified by PCR. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.

Explain Synteny.

-Sometimes even the order of genes on a chromosome is conserved over large segments of the genomes of closely related species. Synteny is a conserved gene order that provides additional evidence for an orthologous relationship between genes at identical physical locations on the same chromosome within the related segments. Sometimes the order of genes on a chromosome is conserved over large segments of the genomes of closely related species. Large segments of the mouse and human genomes have synteny.

Describe the backbone structure of DNA. What is the charge of a polynuceotide?

-Successive nucleotides of both DNA and RNA are covalently linked through phosphate-group "bridges," in which the 5' phosphate group of one nucleotide unit is joined to the 3' hydroxyl group of the next nucleotide creating a phosphodiester linkage. Each linkage is in the same orientation in the chain (5' to 3': 5' end lacks a nucleotide at the 5' position and the 3' end lacks a nucleotide at the 3' position). -Covalent backbones of nucleic acids consists of alternating phosphate and pentose residues, and the nitrogenous bases may be regarded as side groups joined to the backbone at regular intervals -DNA has two strands that run anti-parallel to each other -purine and pyrimidine bases are hydrophobic -DNA double helix is held together by H bonding btwn complementary base pairs and base-stacking interactions. The complementarity between the DNA strands is attributable to the H bonding between base pairs. The base stacking interactions, largely nonspecific with respect to the identity of the stacked base, make the major contribution to the stability of the double helix. -The charge of the backbone is negative (DNA and RNA); also hydrophilic. The hydroxyl groups of the sugar residues form H bond with water. The phosphate groups, with a pKa near 0, are completely ionized and negatively charged at pH 7, and the neg charges are generally neutralized by ionic interactions with positive charges on proteins, metal ions, and polyamines

How are the bases geometrically arranged in relationship to the ribose sugar in a polyncleotide?

-The bases are perpendicular to the ribose sugar in a polynucleotide. This creates a structure where the bases are facing inwards (bases are hydrophobic) -Hydrophobic stacking interactions in which two or more bases are positioned with the planes of their rings parallel (like a stack of coins) are one of the two important modes of interaction between bases in nucleic acids. The stacking also involves a combination of van der Waals and dipole-dipole interactions between the bases -Base stacking helps to minimize contact of the bases with water, and base-stacking interactions are very important in stabilizing the 3D structure of nucleic acids -The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis.

How do we sequence DNA?

-Sangar and the Maxam-Gilbert Method. In both methods, the general principle is to reduce the DNA to four sets of labeled fragments. Fragments are amplified (PCR) then fragments are separated on a gel by electrophoresis so the lengths of the fragments correspond to positions in the DNA sequence where a certain base occurs. The Sanger method (which uses fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method) is used most often and requires the enzymatic synthesis of a DNA strand complementary to the strand under analysis. -to reduce the DNA to four sets of labeled fragments. The reaction producing each set is base-specific, so the lengths of the fragments correspond to positions in the DNA sequence where a certain base occurs. Because the fragments are radioactively labeled at their 5' ends, only the fragment to the 5' side of the break is visualized. When the sets of fragments corresponding to each of the four bases are electrophoretically separated side by side, they produce a ladder of bands from which the sequence can be read directly. -Sanger method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs), the latter of which terminate DNA strand elongation. These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between the two nucleotides, causing DNA polymerases to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labeled for detecting in automated sequencing machines. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides while three other nucleotides are ordinary ones. Putting this in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. Following rounds of template DNA extension fro the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C). The DNA bands may be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image. (when looking at gel, the dark band indicated a DNAfragment that is the result of chain termination after incorporation of ddATP, ddGTP, ddCTP, or ddTTP. The relative positions of the bands among the four lanes are then used to read the DNA seq.) In the automated Sanger reaction, primers are used that are labeled with four different coloured fluorescent tags. PCR reactions, in the presence of the different dideoxy nucleotides, are performed as described above. However, next, the four reaction mixtures are then combined and applied to a single lane of a gel. The colour of each fragment is detected using a laser beam and the information is collected by a computer which generates chromatograms showing peaks for each colour, from which the template DNA sequence can be determined. -The Maxam-Gilbert method is based on nucleotide-specfic cleavage by chemicals and is best used to sequence oligonucleotides (short nucleotide polymers, usually smaller than 50 base-pairs in length). The Maxam-Gilbert method takes a DNA fragment that has been labeled at the 5'-end, usually with radioactive phosphorus, and subjects to chemical cleavage with reagents that cut at specific bases. By using a low concenrtation of the reagents, only one or two sites are cleaved on each molecule. The products produced by cleavage are separated by electrophoresis and the bands made with the different reagents are run side by side and compared. Since only fragments with the labeled 5'-end are visible, the size of the fragment says that a site sensitive to that reagent (a specific base) is located x nucleotides from the 5'-end, again allowing the seqience to be read off.

Please you should be able to draw the nucleotides of DNA and RNA. And given a series of bases draw a DNA or RNA.

-base of nucleotide is joined covalently (N-1 of pyrimidines and N-9 of purines) in an N-B-glycosyl bond to the 1' C of the pentose and the phosphate is esterified to the 5' C. -purine: Adenine and guanine -pyrimidines: Cytosine, thymine (DNA), and uracil (RNA)

What exactly is reveres transcriptase? (hint this is a nickname)?

Full name: "RNA dependent DNA polymerase" Reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. RT is needed for the replication of retroviruses (e.g., HIV). RT activity is also associated with the replication of chromosome ends (telomerase) and some mobile genetic elements (retrotransposons). They are also used in making cDNA libraries out of mRNA. -creates single-stranded DNA from an RNA template. The process of reverse transcription is extremely error-prone and it is during this step that mutations may occur.

Describe the three forms of the DNA molecule. (There similarities and differences structurally.) Is RNA helical?

B form: -the most stable structure for a random sequence DNA molecule under physiological conditions; it is the standard point of interest in the study of properties of DNA. -It is arranged in a right-handed double helix -with 10.5 base pairs per helical turn. -Plane of the base pairs are perpendicular to the helix axis. -Major and minor grooves are present. -There is a 2'endo pucker. A form: -favored in many solutions that are devoid of water. -It is still arranged in a right-handed double helix, but the helix is wider -with 11 base pairs per helical turn. -The plane of the base pairs is tilted 20 deg. relative to the B form: deepens the major groove while making the minor groove shallower -Major and minor grooves present. -There is a 3'endo pucker. Z form: -Arranged in a left-handed helical rotation -with 12 base pairs per helical turn. -The structure appears more slender and elongated; -the backbone takes on a zigzag appearance. -Certain nucleotide sequences fold into Z form much more readily -To form Z form, the purine residues flip to the syn conf., alternating with pyrimidines in the anti-conformation. -major groove is barely apparent in Z-DNA, and the minor groove is narrow and deep

From our lecture explain the concept of Alignment (nucleotides and amino acids)? What can we learn about a genes/protein from alignments? 36. Where does the information needed for an alignment about a particular gene come from? (this came from lecture).

-The electronic search process can be thought of as sliding one sequence past the other until a section with a good match is found. Within this sequence alignment, a positive score is assigned for each position where the amino acid residues in the two sequences are identical—the value of the score varying from one program to the next—to provide a measure of the quality of the alignment. The process has some complications. Sometimes the proteins being compared match well at, say, two sequence segments, and these segments are connected by less related sequences of different lengths. Thus the two matching segments cannot be aligned at the same time. To handle this, the computer program introduces "gaps" in one of the sequences to bring the matching segments into register. Of course, if a sufficient number of gaps are introduced, almost any two sequences could be brought into some sort of alignment. To avoid uninformative alignments, the programs include penalties for each gap introduced, thus lowering the overall alignment score. With electronic trial and error, the program selects the alignment with the optimal score that maximizes identical amino acid residues while minimizing the introduction of gaps. -Sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. In sequence alignments of proteins, the degree of similarity between amino acids occupying a particular position in the sequence can be interpreted as a rough measure of how conserved a particular region is among lineages. The absence of substitutions, or the presence of only very conservative substitutions in a particular region of the sequence, suggests that this region has structural or functional importance. -EuPathDB- this has info about all of the human pathogens. -Can go to amoebaDB, put in a gene and get alignment data. -Compare the sequence to every one of the other protozoans and it gives you data about the sequences. -You can find where you locate the gene in the chromosome, -you can find its predicted protein seq, its homologies, its RNA seq, its genomic seq. NCIB - look at the NIH genomic library, 1000's of organisms. BLAST - can take a copy of the genomic sequence and compare to other organisms. You then get data alignment.

Why is there a major and minor groove formed in the double helix of DNA?

-The offset pairing of the two strands creates a major and minor groove on the surface of the duplex. The strand backbones are closer together on one side of the helix than on the other. The major groove occurs where the backbones are far apart where protein can bind, the minor groove occurs where they are close together, where water binds. -The major and minor grooves result from the overall geometry of the Watson-Crick base-pair. It you look at an A:T, T:A, G:C, or C:G base pair, you will notice that the glycosidic bonds are in about the same place in all base-pairs, and that they are oblique with respect to one another. If you extend the glycosidic bond in each base-pair to form an imaginary line, the lines will form an obtuse and an acute angle. The major and minor grooves form between the phosphate backbones of the DNA molecule, so that the obtuse angle forms the major groove, and the acute angle forms the minor groove.

Describe the 'puckering' of the ribose sugar. Explain syn and anti structures based on rotation.

-The pentose ring is not planar but occurs in one of a variety of conformations generally described as puckered. -Ribofuranose rings in nucleotides can exist in 4 different puckered conformations. In all cases, four of the five atoms are in a single plane. The fifth atom (C-2' or C-3') is on either the same (endo) or the opposite (exo) side of the plane relative to the C-5' atom. -Because of steric constraints, purines in purine nucleotides are restricted to two stable conformations with respect to deoxyribose, called syn and anti. Pyrimidines are generally restricted to the anti conformation because of steric interference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine -The conformation of a nucleotide in DNA is affected by rotation about 7 different bonds. Six of the bonds rotate freely. The limited rotation about bond 4 gives rise to ring pucker, in which one of the atoms in the 5-membered furanose ring is out of the plane described by the other four (endo or exo). -Without hinderance from groups on either ring, the adenine or guanine ring can rotate about the N-glycosidic bond (Carbon1), and form the syn conformation (angle near 0 deg) or anti conformations (angle near 180). Adenine and guanine can rotate between the anti and syn conformations, but the anti is favored in DNA, while syn is favored in the free nucleotide. Thymine and Cytosine occur only in the anti conformation.

From the WORPRESS articles on miRNA and siRNA, how might you employ these experimentally?

-To specifically blockmRNA of interest: siRNA will degrade it and miRNA will inhibit its translation. -Knockout genes that are needed for development. Transform cells with construct containing sequence for hairpin RNA. RNA forms hairpin goes through processing to silence target gene. Place location specific promoter to express in certain cells or constitutively active promoter, to express at all times during development. Problems arise when target gene is needed for correct development. In that case, inject target cells with dsRNA with sequence of interest. Cell will produce siRNAs in response and knockdown expression of target gene. The problem here is that this mechanism is transient. Requires that constant stream of dsRNA is provided so that cell can make adequately silence target gene.

Explain T-T dimerization? Could this cause a disease? (hint what body parts are exposed to UV radiation???

-Ultraviolet light is absorbed by a double bond in thymine and cytosine bases in DNA. This added energy opens up the bond and allows it to react with a neighboring base. If the neighbor is another thymine or cytosine base, it can form a covalent bond between the two bases. These dimers are awkward and form a stiff kink in the DNA. This causes problems when the cell needs to replicate its DNA. DNA polymerase has trouble reading the dimer, and considers it as a single base, since it doesn't fit smoothly in the active site. This causes a frame shift mutation (the worst kind of mutation): if this happens to be in an important gene that controls the growth of cells, such as the genes for Src tyrosine kinase or p53 tumor suppressor, the mutation can lead to cancer (melanoma).

How do we employ this unique sequence driven enzyme of bacteria?

-Very useful tool for research, cloning, DNA digestion. To be able to clone a DNA insert into a cloning vector, both DNAs have to be treated with two restriction enzymes that create compatible ends. -cut plasmid in two spots with restriction enzymes (seq specific) and then take those same two restriction enzymes cut the eukaryoyic chromosome. Then piece of DNA is put into plasmid with DNA ligase (forms new phosphodiester backbone/bond). The recombinant vector is introduced into the host cell (can be done with heat shocking). The host cell is going to reproduce with the plasmid inside

How do we engineer the expression of a protein to aid in affinity purification of that protein?

-We engineer genes to be overexpressed by placing a constitutively active promoter prior to the target gene. Genes are overexpressed to create a surplus of protein product making it easier to isolate proteins that are normally not produced in isolatable concentrations. Recombinant proteins can be tagged for purification The tag binds to the affinity resin, binding the protein of interest to a purification column. -Proteins can be modified to contain a high-affinity tag. The tag binds to the affinity resin, binding the protein of interest to a purification column.

What is a SNP? SSRs? CONTIG?

-Within the human population are millions of single-base differences, called single nucleotide polymorphisms, or SNPs. Each human differs from the next by about 1 bp in every 1,000 bp. From these small genetic differences arises the human variety we are all aware of—differences in hair color, eyesight, allergies to medication, foot size, and even (to some unknown degree) behavior. Some of the SNPs are linked to particular human populations and can provide important information about human migrations that occurred thousands of years ago and about our more distant evolutionary past. -3% or so of the human genome consists of highly repetitive sequences, also referred to as simple-sequence DNA or simple sequence repeats (SSR). These short sequences, generally less than 10 bp long, are sometimes repeated millions of times per cell. Studies suggest that simple-sequence DNA does not encode proteins or RNAs. Unlike the transposable elements, the highly repetitive DNA can have identifiable functional importance in human cellular metabolism, because much of it is associated with two features of eukaryotic chromosomes: CEN and TEL -CONTIG: a set of overlapping DNA segments that together represent a consensus region of DNA. Contigs can thus refer both to overlapping DNA sequence and to overlapping physical segments (fragments) contained in clones depending on the context. A sequence contig is a contiguous, overlapping sequence read resulting from the reassembly of the small DNA fragments generated by bottom-up sequencing strategies. Bottom-up DNA sequencing strategy involves shearing genomic DNA into many small fragments, sequencing these fragments, reassembling them back into contigs and eventually the entire genome.

Compare two well-established plasmid; pBR322 and YAC. How are they similar? Different? Why does Goldberg use TOPO pCR2.1 or pCR4.0 instead of overnight PCR? (from lecture) Why remove the fragment from TOPO and clone it into pQE32?

-YAC contains an ORI, restriction sites, a CEN, two TEL, and two selectable markers. pBR322 contains an ORI, restriction sites, and ampicillin/tetracycline-resistant genes. YACs are high-capacity cloning vectors and can hold larger genomes that pBR322. Yeasts are eukaryotic and, therefore, better than pBR322 vectors for the expression of eukaryotic genes. Both YAC and pBR322 are very easy to maintain and grow in the laboratory. Both YACs and pBR322 are constructed under the same principles (origin of replication, specialized sequences, small size). Both YAC's and pBR322's entire sequence is known. YAC is extremely large and pBR322 is way smaller and can reproduce much quicker. -pQE32: contains IPTG-inducible promoter and 6xHis Tag for purification or identification -The TOPO's vector has 3' T overhangs, which are complementary to the PCR product A overhangs. The plasmid also has topoisomerase enzyme linked to its 3' end for fast ligation of the PCR product in the vector. This is much more efficient than typical ligation reactions (overnight PCR) which take a long time.

Explain secondary structure in DNA and RNA. What are palindromes? Hairpins and cruciforms?

-palindrome: common type of DNA sequence. The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA. Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform (cross-shaped) structures When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat. Mirror repeats do not have complementary seqs within the same stand and cannot form hairpin or cruciform structures. To superimpose one repeat (shaded sequence) on the other, it must be rotated 180˚ about the horizontal axis then 180˚ about the vertical axis -cruciforms: making a cross with holiday junctions. Cruciform DNA structure appears as several hairpin loops, creating a crucifix-like structure composed of double-stranded DNA. DNA structure is formed by incomplete exchange of the strands between the double-stranded helices. -Hairpin loops are formed by a fold in a single strand of DNA or RNA, causing several bases to remain unpaired while the strand loops back upon itself. Hairpin loops form between nearby self-complementary sequences. Resulting hairpins are more common type of secondary structure in RNA. Short base sequences (like UUCG) are often found at the ends of RNA hairpins and are known to form tight and stable loops. The 2'-hydroxyl group of ribose can H-bond with other groups. -RNA has no simple, regular secondary structure that serves as a reference point, as does the double helix for DNA. The three-dimensional structures of many RNAs, like those of proteins, are complex and unique. Weak interactions, especially base-stacking interactions, play a major role in stabilizing RNA structures, just as they do in DNA. Where complementary sequences are present, the predominant double-stranded structure is an A-form right-handed double helix. Hairpin loops form between nearby self-complementary sequences. The potential for base-paired helical structures in many RNAs is extensive (Fig. 8-27), and the resulting hairpins are the most common type of secondary structure in RNA.

What is a plasmind? How was a plasmid constructed? What was used before plasmids? What is a Transposable Element?

-plasmids are circular DNA molecules that replicate separately from the host chromosome. They can be introduced into bacterial cells by a process called transformation. The cells (generally E. coli) and plasmid DNA are incubated together at 0 ˚C in a calcium chloride solution, then sub- jected to a shock by rapidly shifting the temperature to 37 to 43 ˚C. For reasons not well understood, some of the cells treated in this way take up the plasmid DNA. Some species of bacteria are naturally competent for DNA uptake and do not require the calcium chloride treatment. In an alternative method, cells incubated with the plasmid DNA are subjected to a high-voltage pulse. This approach, called electroporation, transiently renders the bacterial membrane permeable to large molecules. -Requirements: (1) Origin of replication (ORI). They must be able to replicate themselves or they are of no practical use as a vector. (2) Selectable marker. They must have a marker so you can select for cells that have your plasmids. (3) Restriction enzyme sites in non-essential regions. You don't want to be cutting your plasmid in necessary regions such as the ORI. (4) Small. If they are small, they are easier to isolate (you get more), handle (less shearing), and transform. -A transposable element (TE or transposon) is a DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell's genome size. Transposition often results in duplication of the TE. -LAMDA GENOME was used before plasmids. (easy to turn on and off)

Do explain the functions of the following enzymes as technological tools in clone, transforming and expressing genes; DNA ligase, Alkaline Phosphotase, Terminal transferase, polynucleotide kinase, reverse transcriptase, Exonuclease and Restriction Endonucleases.

DNA ligase: a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of new phosphodiester bonds in a reaction that uses ATP or a similar cofactor. It plays a role in both DNA replication (DNA ligase I) and DNA repair(DNA ligase III and IV). Also, purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA. With this, you can put a gene of interest in a plasmid. Alkaline Phosphatase: a hydrolase enzyme responsible for removing terminal phosphate groups from the end of the DNA of many types of molecules, including nucleotides, proteins, and alkaloids. So if you add the alkaline phosphates, it removes the phosphates so the vector will wait for an insert to ligate and you will have the clone/vector with the insert you want. **Prevents the circular plasmid from closing in on itself before the gene of interest is inserted. Terminal Transferase: catalyzes the addition of deoxynucleotides (specifically, homopolymer tails) to the 3'-OH ends of a DNA strand. Unlike most DNA polymerases, it does not require a template (unlike DNA polymerase) so it can add any nucleotide you like at the 3' end. It can be useful for labeling DNA molecule (with radioactivity) or add a homopolymeric tail to a DNA molecule (poly A tail for example) that can then be amplified with a primer containing a bunch of Ts. Polynucleotide Kinase: catalyzes the addition of a phosphate to the 5'-hydroxyl end of a polynucleotide to label it or permit ligation. **Opposite activity as Alkaline Phosphatase. Once you want the plasmid it to close around an insert. Still need DNA ligase. Reverse Transcriptase: generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Scientists often generate cDNA libraries from mRNA as a way to find genes of interest. They screen these libraries using what are known as probes--complementary pieces of DNA that hybridize to the cDNA molecules. They also use cDNA libraries to identify genes that are expressed differently in different types of tissues or at different developmental stages. Exonuclease: enzymes that work by catalyze the breakage of phosphodiester bonds at either the 3' or the 5' end occurs. The T5 exonuclease chews back DNA from the 5' end. The resulting single-stranded regions on adjacent DNA fragments can anneal, which then allows the DNA polymerase to incorporate nucleotides to fill in any gaps. Then, the Taq ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA.The entire mixture is incubated at 50°C for up to one hour. If in a plasmid, the resulting product can be directly transformed into E. coli. (removes nucleotide residues from the 3' ends of a DNA strand) Restriction Endonuclease: enzymes that recognize and cleave foreign DNA (the DNA of an infecting virus, for example) at specific nucleotide sequences, usually 4-6 nucleotides long with both strands complementary to one another. To be able to clone a DNA insert into a cloning or expression vector, both DNAs have to be treated with two restriction enzymes that create compatible ends.

Explain what is a Restriction Endonuclease. How might these enzymes work in eukaryotic cells?

-recognize and cleave DNA at specific DNA sequences (recognition sequences or restriction sites) to generate a set of smaller fragments -Three types: Types I and III are generally large, multisubunit complexes containing both the endonuclease and methylase activities. Type I cleave DNA at random sites that can be more than 1000 base pairs from the recognition sequence. Type III cleave the DNA about 25 bp from the recognition sequence. Both types move along the DNA in a rxn that requires the E of ATP. Type II require no ATP, and cleave the DNA within the recognition sequence itself. The recognition sequences are usually 4-6 bp long and palindromic. -sticky ends: some of these make staggered cuts on the two DNA strands leaving two to four nucleotides of one strand unpaired at each resulting end. They can base pair with each other or with complementary sticky ends of other DNA fragments -Blunt ends: other cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends -Their biological function is to recognize and cleave foreign DNA; such DNA is said to be restricted. In the host cell's DNA, the sequence that would be recognize by its own restriction endonuclease is protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase. In eukaryotes, RE (which is actually called just endonucleases) prevents DNA damage from environmental agents and prevent the supercoiling of DNA at replication forks, by cutting the backbone, relieving the tension and pasting the ends together again. -DNA fragment to be cloned can be joined to a suitable cloning vector by using DNA ligases to link the DNA molecules together. The recombinant vector is then introduced into a host cell, which amplifies the fragment in the course of many generations of cell division.