BCH5413 Exam 1

What is the difference between euchromatin and heterochromatin?

Euchromatin: Found more towards the center of the nucleus. Lightly stained, more accessible DNA (not compacted), transcriptionally active. Heterochromatin: Found closest to the nuclear membrane. Darkly stained, closed inaccessible DNA (very compacted), transcriptionally inactive, higher levels of chromatin organization. DNA can be arranged differently within the same nucleus. All cells have different levels of organization of their DNA but what's in them is different from each other depending upon the function of that cell.

What is the meaning of the term exon shuffling? How is this process related to the development of new proteins?

Exon shuffling is a molecular mechanism for the formation of new genes. It is a process in which two or more exons from different genes can be brought together ectopically or the same exon can be duplicated to create a new exon-intron structure. Exons can be shuffled and create different combinations to give you different types of proteins. Some exons correspond to protein functional domains. This can happen even if the proteins are part of the same family member. Two proteins can have different functions but share some exons. These are homologous regions.

True/False: Blunt ends gives better ligation than sticky ends

False

True/False: If bacterial cells have taken up the plasmid, the colonies are white in blue/white selection.

False

True/False: Chargaff's rule: A=G and T=C

False The rule is that DNA from any organism should have a stoichiometric relationship of 1:1 for pyrimidines : purines or stated mathematically C + T (pyrimidines) = A + G (purines). The number of pyrimidines (C, T) however does not need to be equal (C=T), or purines for that matter.

True/False: Most biological DNA is positively supercoiled.

False, Biological DNA is negatively supercoiled

True/False: GC rich DNA has a lower melting temperature than AT rich DNA

False, GC rich DNA has a higher melting temperature because it forms 3 hydrogen bonds compared to the 2 hydrogen bonds by AT.

What is a GST tag? How does it help in protein purification?

Glutathione-S-transferases (GST) are a family of multifunctional cytosolic proteins that are present in eukaryotic organisms. Because the isoforms of these proteins are not normally found in bacteria, there are no endogenous bacterial proteins that can outcompete GST-fusion proteins for binding in protein purification. The GST tag is used as an affinity tag to enhance solubility of many eukaryotic proteins expressed in bacteria and it is based on its' strong affinity for immobilized glutathione-covered matrices. GST is one of the solutions used in protein-protein interactions using fusion proteins as a solution if a protein is insoluble.

In DNA, C2 is bonded to... This is important because...

H Importance - C2 does not have a hydroxyl group, which distinguishes it from RNA.

Linker Histone

H1

Core Histones

H3, H4, H2A, H2B

In the analysis of Per1 protein expression: What is HRP and why is it important in the western blot process?

HRP is horseradish peroxidase, an enzyme that has been linked to the secondary antibody. The substrate to the HRP enzyme is added to the reaction, and interaction between these two results in light and detection via chemiluminescence.

Heterozygous

Having inherited different versions (alleles) of a genomic marker from each biological parent. An individual who is heterozygous for a genomic marker has two different versions of that marker.

Give a brief summary of how the IPTG induction system works.

IPTG induces the expression of genes under the control of the lac operon. IPTG works by removing a repressor (LacI) from the lac operon, which then allows RNA polymerase to bind to the promoter and begin transcription, and therefore expression.

What are the steps to be taken if the protein is insoluble?

If a protein is insoluble, one should first collect the inclusion bodies and reform the protein. Reducing the growth temperature could also help increase the solubility of the protein. Another consideration would be to use a low or moderate copy number plasmid vector such as PBR322 to help increase solubility. If the protein continues to be insoluble, one can fuse a periplasmic targeting sequence to the N-terminus. Another fusion option would be to use a fusion protein such as glutathione S transferase. If all these options fail to help increase solubility, then it would be best to find a new protein with a higher solubility.

5. What does the Meselson and Stahl experiment confirm?

In 1958 Meselson and Stahl published their experiment of DNA replication in Escherichia coli. In this experiment they started their DNA molecule with heavy nitrogen bases and when they are separated the new strand made was with light nitrogen bases and the new daughter strand each was found to have one heavy and one light strand which proved the DNA replication mechanism as semiconservative DNA replication model as proposed by Watson and Crick.

Describe how transfection of plasmids into mammalian cells is different from transformation of plasmids into bacterial cells.

In mammalian cells, transfection is lipid-mediated in which DNA is incubated with lipofection reagent to form a DNA lipid complex

Why might a researcher choose an inducible vector?

In some experiments, overexpressing proteins of interest can be toxic to the cells, so scientists can keep a cloned gene repressed until expression is needed.

Why would you do site directed mutagenesis?

Introduce or remove tags for purification or identification Use to study the relationship between protein structure and function Screen for mutants that have desired characteristics Study transcription factor binding sites

What is the difference between and intron and an exon?

Introns are non-coding sequences present in DNA and are removed before translation begins through RNA splicing. On the other hand, exons are gene coding sequences of DNA. Unlike introns, exons are present in mRNA.

In Maxam & Gilbert Sequencing: Why is this method considered direct as compared to other methods?

It is Directly sequenced because it does not need a primer and the sequence is read by cutting DNA up into fragments that they can read. Other methods sequence DNA by annealing a primer to the DNA sequence so that you can create a new sequence that is complimentary base paired to the sequence that you want to know.

Why do you need a loading control for a northern blot? What would be an ideal loading control for a northern blot?

Loading control needed to ensure equal loading of RNA in all lanes. To compare changes in RNA expression levels, need to load same amount of RNA for both the control and the treated condition. A common control is rRNA.

Why do you need a loading control for a western blot? What would be an ideal Loading control for a western blot experiment?

Loading control needed to ensure equal loading of sample in all lanes. Ideal controls for western blotting are housekeeping proteins such as actin or GAPDH, proteins whose expression should not change with the hormone treatment.

In a PCR reaction, why do you need: Magnesium?

Magnesium is required as a co-factor for thermostable DNA polymerase. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.

What is the main advantage of microarray technology over QPCR?

Microarrays allow visualization of every expressed gene in a cell at one time while QPCR allows to look at changes in gene expression one gene/mRNA at a time.

What is the basic difference between measuring RNA expression by a Northern blot compared to using In situ hybridization?

Northern blot measures RNA expression by doing gel electrophoresis of an RNA sample and then transferring to a membrane via Northern transfer. In situ hybridization measures RNA expression in a cellular or tissue location.

In DNA, C3 is bonded to... This is important because...

OH Importance - The C3 of one sugar forms a phosphodiester bond with the 5` phosphate of another sugar.

How can one stretch of DNA be used to create more than one protein?

Once the DNA is transcribed to RNA, the introns are removed and some exons may be removed as well through a process known as splicing. Alternate splicing can give you different protein combinations due to the exons that are present, for example one protein can contain exon 1, 2, and 3 while another contains exon 1, 3 and 4. The protein that is created depends on the regulation of the splicing mechanism and what type of mechanism is being used.

Pull-down assay to study protein-protein interactions

One method used to study protein-protein interaction is the Pull-down assay. Pull-down assays can be used to study a known protein interaction or to determine if an unknown one exists. Is this assay, a protein which can bind to the desired protein of study is added to a solution to act as "bait." It is then bound to a specific compound that will limit its binding of other molecules that aren't our protein of interest. The new complex is washed to remove all unbound proteins and placed in a solution containing the protein of interest. Once the desired protein has bound to the bait complex, a second wash occurs to again wash away all unbound proteins. The complex is then eluted using a method specific to the original protein and binding compound. Once only the protein-protein complex remains, its run through SDS-PAGE to determine what the interaction is.

In genotype analysis of a mouse knockout experiment, describe how the two different forward primers with one reverse primer would allow you to distinguish between a mouse that is heterozygous, homozygous normal (no knockout), and homozygous knockout.

PCR can be used to identify if a mouse is a KO or not. How a KO mouse is generated: using a targeting vector. Take a sequence (in Dr. Gumz experiment, she used PGK-Neo) and use it to replace a part of the gene that you want to KO (Dr. Gumz KO exons 10, 11, and 12 from the Per1 gene in mouse kidney cells). To identify if a mouse is heterozygous, homozygous normal (WT), or homozygous KO, three primers are used: Two forward primers: WT forward primer anneals to the normal gene (Dr. Gumz used a forward primer that anneals to exon 10). KO forward primer anneals to the sequence that has been KO (Dr. Gumz used a forward primer that anneals to endogenous sequence PGK-Neo). One reverse primer that anneals to a portion of exon 13, which should be present in all three genotypes.

In a PCR reaction, why do you need: DNA polymerase?

PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates.

ChIP: How would an investigator determine if a specific DNA sequence is bound to the protein of interest?

PCR using gene-specific primers to detect DNA of interest, positive product confirms protein pulled was bound to DNA sequence of interest The protein isolate from the immunoprecipitation assay is degraded and PCR is performed using primers specific to the DNA sequence of interest. The PCR product is then evaluated using agarose gel electrophoresis. A positive PCR product suggest that the isolated protein was bound to the DNA sequence of interest.

In the analysis of Per1 protein expression: Why is it important that the secondary antibody is goat anti-rabbit and not rabbit anti-goat?

Primary antibody is rabbit anti-Per 1. Secondary antibody needs to bind to primary antibody. Hence, second antibody must be anti-rabbit antibody, made in goat. If the secondary antibody were anti-goat, it wouldn't bind to the primary antibody.

Western Blot Fragment Detection Method

Primary antibody specific for the protein of interest. Protein of interest is on the membrane. Secondary antibody with linked HRP + substrate. If secondary antibody binds primary antibody, light will be produced to indicate presence of protein of interest. Chemiluminescence detection.

In Situ Hybridization

Probe contains colorimetric signal that allows for visual detection. More color = higher presence of RNA of interest.

Northern Blot Fragment Detection Method

RNA of interest is hybridized in the membrane to a radioactively labeled probe. Detection by autoradiography.

What is the main advantage of RNA seq over microarrays?

RNA seq does not rely on a hybridization step like microarrays do. Microarrays having a hybridization step results in the need for other tests to validate results, as hybridization could result in nonspecific binding.

What is a reporter gene? AND, why might it be useful in studying the characteristics of a newly cloned gene?

Reporter genes are genes that help detect either the measurement or level of expression on the gene of interest. This is useful in studying the characteristics of a new gene because it can show or detect the levels of expression on the gene of interest (basically, how the gene is behaving, how it "reacts", and what it's doing). It "reports" the expression levels of the gene.

How can the PCR technique be modified to detect RNA instead of DNA?

Reverse-Transcriptase PCR. Using reverse transcriptase instead of DNA polymerase allows to determine the relative expression level of a gene using RNA instead of DNA as the initial sequence. How it works (quick overview): Use mRNA as initial sequence, add oligo dT (primer that recognizes/hybridizes to the poly-A tail of mRNA) and reverse transcriptase to synthesize the first strand of DNA, resulting in a double-stranded hybrid of mRNA and DNA. RNAse H degrades some of the mRNA, which serve as primers to begin synthesis of the second strand. DNA polymerase extends these primers. End result: ds-cDNA - ds complementary DNA that can be used as template in a subsequent PCR reaction.

In gel electrophoresis, why does DNA move according to size?

Smaller DNA fragments will move faster towards the positive pole when compared to larger DNA fragments. Hence, smaller DNA fragments will travel a longer distance than larger fragments and these will be separated according to size.

In Maxam & Gilbert Sequencing: Why do you need to chemically modify the bases?

So that the DNA can be cleaved at specific bases

What is the basic difference between a Southern, a northern, and a western blot? (ie. What does each detect?)

Southern blot detects DNA, agarose gel to nylon membrane. Northern blot detects RNA, agarose gel (containing formaldehyde) to nylon or nitrocellulose membrane. Western blot detects proteins, polyacrylamide gel (containing SDS and b-ME) to membrane.

In Southern blot analysis: What is the meaning of the term stringency?

Stringency: properties of hybridization conditions that dictate the degree of base mismatch permitted between probe and target DNA.

How has laundry detergent been improved by SDM?

Subtilisin is a protease used in laundry detergent. Bleach is able to oxidize the methionine group and render the protease inactive. By using SDM, methionine is mutated to alanine and the protease remains active.

Give two advantages of TaqMan probe over Sybr Green.

TaqMan probe allows for specific hybridization, while the SYBR green I dye binds to dsDNA indiscriminately (non-specifically) and need other tests to confirm results. More than one TaqMan probe can be designed to amplify two distinct sequences in one reaction tube. SYBR green binds indiscriminately to all dsDNA and so this is not possible.

After isolating intact chromatin from cells, lightly digesting it with micrococcal nuclease and running it on an agarose gel, you see a banding pattern of repeating 200bp fragments (200, 400, 600 etc). Explain this result.

The 200bp banding pattern is showing that the micrococcal nuclease has cut between each individual nucleosome. The 400bp pattern is showing that the nuclease has cut between every 2 nucleosomes and the 600bp pattern is every 3 nucleosomes, etc. This experiment was performed to show that parts of DNA are protected from the nuclease by nucleosomes. When the concentration of nucleus increases, there are more individual nucleosomes. Nucleases one are 200bp each 200bp fragments= individual nucleosome

Describe why DNA has directionality.

The 5' end has a free phosphate group and 3' end has a free hydroxyl (OH) group. Nucleotide strands form hydrogen bonds between the nitrogenous base off the 1' Carbon. DNA is always synthesized in the 5'-to-3' direction, which means that nucleotides are added only to the 3' end of the growing strand. In DNA, G forms 3 hydrogen bonds to C, A forms two hydrogen bonds to T. Sequence of DNA has an effect on the structure.

How is DAPA different from ChIP for determining if a protein is bound to a specific DNA sequence?

The ChiP assay allows analysis of DNA- protein interaction in living cells by treating with formaldehyde

What are the advantages of RNA-seq over microarrays?

The advantages of RNA-seq over microarrays are that RNA-seq has higher sensitivity and higher dynamic range than microarrays, novel transcripts sequences can be identified, structural variation and alternative splicing can be revealed, and an unlimited amount of sample comparisons can be made.

Compare and contrast the method of base detection between pyrosequencing and ion torrent sequencing.

The base detection methods of pyrosequencing and ion torrent sequencing detect one nucleotide at a time. Additionally, through the breaking of the phosphate bonds, the methods are indirect processes to determine the nucleotide present. For pyrosequencing, if the nucleotide that is flowed across the plate is incorporated as the next nucleotide, then pyrophosphate (PPi) is released. Then, the PPi is broken apart with sulfurylase to produce ATP. The ATP is utilized by luciferase to produce light. The pyrosequencing method detects PPi. For ion torrent sequencing, if the nucleotide that is flowed across the plate is incorporated as the next nucleotide, then Hydrogen ion is released with the breakage of the phosphate bonds. The Hydrogen ion is then utilized to detect a change in pH. The ion torrent sequencing method detects Hydrogen ions.

What is the significance of the fact that histones have an overall basic pH?

The basic pH (positive charge) of the histones attracts the more negative charge of DNA. The interactions between histones and DNA are non-sequence specific. Instead, the interactions between histones and DNA are due to the positive charge of histones and the negative charge of DNA attracting each other.

In cassette mutagenesis, what is the function of T4 PNK AND how does it carry out that function?

The function of T4 PNK is to add a 5' phosphate onto the ends of the oligonucleotides so that they can be ligated together into the DNA

In the experiment described that supports DNA as the genetic material: Why does the mouse die if the researcher mixes the complete dead pathogenic strain with live non-pathogenic strain bacteria and then inject it into the mouse? (The first mouse in the figure).

The mouse dies because the R strain has been transformed and is now an S strain, capable of producing the virulence factors that make the S strain deadly to mice. This is because as the pathogenic strain is killed, its DNA is released, and now floating freely can go into the R strain and transform it.

Initially investigators believed that the nucleolus was an organelle within the nucleus of some cells. Explain why that is no longer considered to be accurate.

The nucleolus is a region found within the nucleus that stains dark, like heterochromatin, but caused by the highly active transcription occurring in this area. This is also where ribosomal RNA is produced. The nucleolus has a unique density that separates it from its surrounding nucleoplasma, making it a separate compartment without the need of a membrane.

In the experiment described that supports DNA as the genetic material: What was the only scenario in which the mouse lives? Why?

The only scenario in which the mouse lives is when DNA is destroyed. This is because it's DNA that contains the genetic information that codes for the virulence factors that kill mice when they become infected.

In Sanger Sequencing: Why do you need a primer?

The primer in Sanger sequencing anneals to the template ssDNA, DNA polymerase will bind and extend the primer, adding dNTPs and ddNTPs. It is vital to know some of the sequence of the targeted DNA so that a primer can be utilized to make the complementary DNA.

In a PCR reaction, why do you need: Both a forward and a reverse primer?

The purpose of PCR primers is to provide a "free" 3'-OH group to which the DNA polymerase can add dNTPs

In a PCR reaction, why do you need: dNTP?

The purpose of the deoxynucleotide triphosphates (dNTPs) is to supply the "bricks." Since the idea behind PCR is to synthesize a virtually unlimited amount of a specific stretch of double-stranded DNA, the individual DNA bases must be supplied to the polymerase enzyme.

In the EMSA technique, how would an investigator know if a protein is bound to a specific DNA sequence?

The radioactively labeled primer set (bare DNA) is incubated with the protein of interest, and if there is interaction between the primers and the DNA-binding protein then the resulting complex will not travel as far on the gel (it will be higher up) compared to just the bare DNA. If this higher band is seen on the gel, then the investigator would know that the protein is bound to the specific DNA sequence.

In a PCR reaction, why do you need: Template?

The template DNA is required so that more copies of that DNA can be made

What value was there in the discovery and then use of Thermus Aquaticus DNA polymerase in PCR? Why did it make the process more efficient, describe specific steps in the process?

Thermus Aquaticus DNA polymerase is heat stable. When PCR was done before this discovery, more polymerases had to be added with each cycle. The discovery of this thermostable polymerase allowed for automation of PCR method.

In Maxam & Gilbert Sequencing: Why do you need to radioactively label the fragment that is being sequenced?

To be able to read the sequence on the acrylamide gel

Bait and Prey Method to study protein-protein interactions

To study protein-protein interactions you can use the "bait and prey" method with fusion proteins. First you have to immobilize the first "bait" protein to agarose beads then wash away any unbound protein. Then you have to add your "prey" protein and let them incubate and interact before washing the unbound proteins a second time. You can then elute the proteins that have interacted from the agarose beads and run an SDS-PAGE gel to visualize them. The most common method for visualizing these interactions would be a western blot. From the gel you can see the bands of interacting proteins and bound "bait" proteins .

After any type of mutagenesis is complete, the additional steps of transformation, selection, miniprep, and sequencing are needed. Explain why each is necessary to complete the mutagenesis project.

Transformation: Bacteria uptake genetic material to gain a function, plate the bacteria on media that contains antibiotic, colonies that grow have the plasmid Selection: Pick a colony and grow them in liquid LB (+ selection) overnight to grow more of the bacteria Miniprep: Used to isolate the plasmid Sequencing: In this step we are screening for the mutation in order to check that the mutation actually took effect, usually though sanger sequencing and use a chromatogram, want high signal strength for the new mutation

True/False: E.coli methylate their own DNA to prevent to prevent infection by viruses

True

True/False: Increased salt concentration increases the stability of complimentary base pairing

True

True/False: The general ratio of vector to insert in cloning is 1:3

True

In next generation sequencing, what is the advantage of adapter sequences ligated to DNA fragments?

Ttotal DNA is fragmented, and uniform adapter sequences are ligated to the ends of each fragment. These adapters contain unique sequences that are used to identify each individual sample, which allows the DNA from multiple samples to be pooled and analyzed concurrently. The adapters also contain universal primer recognition sequences such that a single PCR reaction can amplify all sequences. Finally, the adapters contain a sequence that is complementary to a second sequence located on a physical surface—typically a bead or spot on a chip—that enables the library of sequences to be separated spatially for analysis.

What is the difference between a two start supercoiled model and a two start twisted or helical model for the 30nm fiber? Which is more supported by x-ray crystallography data?

Two-start supercoiled model: two nucleosomes at the top that wrap around in a helical form. Two-start twisted/helical model: two nucleosomes at the top but they go one opposite to the other, one nucleosome is opposite to the next. Most predominant structure found in cells: two-start twisted model.

Transformation positive control

Undigested plasmid, you should get a bunch of plasmid that result from transformation

In a western blot, how would you analyze the quality of the protein sample?

Using an example gel and staining it with Coomassie dye to look at the integrity of the samples. If nice, discrete bands are observed, protein sample is of good quality. Also helps to observe if loading was even between samples/lanes. This type of staining can also be done to the membrane itself.

Describe how PCR can be modified to make it quantitative.

Using fluorescence across PCR cycles. As PCR cycles progress, the higher the fluorescent signal is, indicating higher mRNA expression levels. TaqMan Probe-Based Assay: uses a probe with a reporter and quencher. Reporter will only fluoresce when dislodged from the probe and away from the quencher. Dislodging occurs during as DNA pol moves through the probe during amplification. SYBR Green I Dye Assay: this fluorescent binds indiscriminately to dsDNA; hence, as amplification proceeds and more DNA copies are made, the fluorescent signal increases.

After one cycle of PCR, how many DNA fragments to you have that exactly match your target sequence? Explain your answer.

When PCR is started with one dsDNA fragment, there will be two exact copies of the target sequence after one cycle. For each cycle, the PCR doubles the existing number of copies of DNA sequence = logarithmic amplification.

Explain what happens to the 30nm fiber in order to create a metaphase chromosome.

When a cell is ready to divide during mitosis or meiosis, DNA needs to be very compacted so that it can be efficiently transferred. The 30nm fiber is further looped onto a protein scaffold. Scaffolding proteins in the center give the metaphase chromosome its unique shape.

What does it mean to say that a plasmid is transiently transfected into cells?

When saying a plasmid gets transiently transfected into cells, it means that constructed plasmid with the gene of interest is introduced into the cells, but it does not become integrated into the host chromosome. It can be replicated as an episome, meaning it is independent of the host chromosome replication process.

What is the function of IPTG in cloning?

When using expression vectors, cloned genes should remain repressed until its time for expression to prevent toxicity, growth inhibition, and inclusion bodies. The lac promoter is inducible because it remains repressed until stimulated by IPTG

DpnI digestion negative control

Wild-type plasmid that didnt undergo PCR, if colonies from the wild-type form then wild-type plasmid was not fully degraded

Does A DNA or Z DNA have 12 bp per turn?

Z DNA

In DNA, C1 is bonded to... This is important because...

a nitrogenous base (adenine, guanine, cytosine, or thymine) Importance - this allows the nitrogenous bases to form hydrogen bonds (A-T, C-G) between the 5`-3` strand and the 3`-5` strand.

Meiosis

a type of cell division in sexually reproducing organisms that reduces the number of chromosomes in gametes ( the sex cells, or egg and sperm). In humans, body (or somatic) cells are diploid, containing two sets of chromosomes (one from each parent). To maintain this state, the egg and sperm that unite during fertilization must be haploid, with a single set of chromosomes. During meiosis, each diploid cell undergoes two rounds of divisions to yield four haploid daughter cells which are gametes.

ChIP: How is the DNA that is bound to the protein of interest isolated?

add antibody of interest and perform immunoprecipitation to pull the antibody out with the DNA of interest bound to it

Why would it be advantageous for a plasmid vector to have antibiotic resistance gene?

allowing for antibiotic use to distinguish between cells that have taken up the plasmid and those that have not

What is a genomic library?

collection of total genomic DNA for a single organism, coding and noncoding

What is cDNA?

complementary DNA of an RNA

In Sanger Sequencing: Why do you need ddNTPs ?

ddNTPs (dideoxynucleotides) are modified nucleotides that's missing an oxygen on the 3rd carbon . In Sanger sequencing, adding this modified nucleotide will terminate the sequence of chain and no further nucleotide will be added by the DNA polymerase (hence the method being referred to as the chain termination method).

In Quikchange mutagenesis, what is the purpose of dpnI AND how does it carry out that purpose?

dpnI is responsible for targeting the sites within the DNA that are methylated by DAM methylase in vivo. dpnI is going to recognize these methylation sites and chew them up, leaving behind the DNA with the newly incorporated mutation.

Why would it be advantageous for a plasmid vector to have LacI?

encodes for lac repressor protein, particularly important for expression vectors, keeps the clone turned off until the bacteria grows to log phase and it's time for transcription - prevents toxicity, growth inhibition and inclusion bodies

Lower stringency

greater mismatch and higher background noise.

Explain library screening using the plaque hybridization method.

lay nylon membrane on a plate (prepared from packaging step) to pick up DNA from phage, fix, probe membrane with labeled DNA homologous to the sequence of interest, detect by autoradiography, repeat find positive plaques, purify, and inoculate onto another lawn of E. coli to increase the yield of positive plaques

Higher stringency

less mismatch but may result in reduced signal.

With reference to ChIP, how is the chromatin sheared?

mechanically (sonifier) or enzymatically

Ligation negative control

no ligase, no colonies should form on the plate, if colonies form then the plasmid was not digested to completion

In a cloning experiment, what does it mean when you get colonies on a No ligase / No insert control plate? What if there are more colonies there than on your experimental (good or bad? Why?).

no ligase/no insert and no insert are negative controls representing vectors that are self-ligated/undigested. This would be bad because it would mean that very few of the cell took up the plasmid successfully

Transformation negative control

no plasmid, if colonies form then the antibiotics went bad

PCR negative control

no template, if there is a PCR product the reagent is contaminated

In creating a cDNA library what is the advantage of using beads containing oligo T nucleotides for isolating the RNA?

oligo T nucleotides can be used as a primer because they are complementary to the 3' end poly(A) tail of most eukaryotic mRNAs.

Allele

one of two or more versions of DNA sequence (a single base or a segment of bases) at a given genomic location. An individual inherits two alleles, one from each parent, for any given genomic location where such variation exists.

What is the advantage of using a partial digest over a full digest for creating fragments for cloning?

partial digest generates a series of overlapping fragments allowing for the ability to capture the whole gene in a vector, a full digest would only create identical fragments.

In DNA, C5 is bonded to... This is important because...

phosphate Importance - The C5 can be the lead sugar or form phosphodiester bonds with the C3 of a second sugar. This gives DNA the 5` to 3` linkage.

With regard to cloning, what is the significance of the discovery of restriction enzymes?

restriction enzymes are bacterial enzymes that cleave DNA at recognition sequences allowing for DNA of interest to enter plasmid.

Why would it be advantageous for a plasmid vector to have origin of replication?

sequence recognized by host bacteria

Why would it be advantageous for a plasmid vector to have polylinker/mcs?

series of restriction enzyme recognition sites, allows DNA to be put into plasmid

Diploid refers to...

the presence of two complete sets of chromosomes in an organism's cells, with each parent contributing a chromosome to each pair. Humans are diploid and most of the body's cells contain 23 chromosome pairs.

In cDNA library construction, why would an investigator want to create a cDNA library?

to obtain a set of clones representing all mRNAs expressed in a given cell type

Why would an investigator want to create a genomic library?

to study gene regulation, effects of mutations outside coding sequences, and to compare sequences between organisms

Why would it be advantageous for a plasmid vector to have LacZ?

used for blue-white colour selection, encodes alpha B-galactosidase (100aa) the host bacteria encodes carboxyl B-galactosidase (100-1000aa), when combined, they form an active enzyme that releases xanthine dye and galactose upon cleaving X-galactoside (X-gal) - an indicator present in blue-white color section media

With reference to ChIP, how are cells cross-linked?

using formaldehyde In the middle of two regions of closed chromatin there is a region of open chromatin that interacts with protein. The sample is treated with formaldehyde to preserve this protein DNA interaction.

Choosing an E. coli protein expression system has a lot of advantages such as yield, cost and ease. Discuss (not just list) two reasons why a researcher might not choose this type of system. Be specific, what might their goal be and why wouldn't this system work for them?

-An E. coli protein expression system might not be sophisticated enough for the protein we are trying to study, and there could be downstream effects on the protein's expression. An example Dr. Gumz gave was the limitation that E. coli systems cannot support post-translational modifications- so if our protein of interest needs to be methylated or phosphorylated, an E. coli system is not a good match for what we are trying to study.-A second limitation is that there are codon usage differences between E. coli cells and mammalian cells. We might encounter what Dr. Gumz described as "experimental difficulty" when trying to apply findings from an E. coli protein expression system to a mammalian protein expression system, since protein expression may not always be the same

In Illumina sequencing, what are the 2 modifications to Sanger Sequencing ddNTPs AND how are they advantages?

1. Addition of a reversible terminator to the 3' Carbon. The Sanger ddNTP has a Hydrogen at this location which terminates DNA synthesis. In Illumina sequencing, this has been modified to -OR, which is a reversible terminating group. 2. A cleavable linker is added between the nucleotide base and fluorescent dye. Once the fluorescence has been recorded, the dye can be cleaved from the nucleotide and washed away in preparation for sequencing of the next base. In comparison to Sanger ddNTPs, these modifications allow sequencing without the need of DNA fragment separation through a gel. And in combination of the other features of Illumina sequencing, much more DNA can be sequenced in parallel with high accuracy.

Describe similarities between DNA and RNA.

1. Both DNA and RNA are large biological polymers 2. Both DNA and RNA are nucleic acids 3.Both DNA and RNA consists of sugar, nitrogenous bases, and a phosphate backbone 4. On both molecules Guanine pairs with Cytosine 5. Both store genetic information

What are the 2 advantages of PacBio sequencer?

1. The fluorophore attached to the phosphate gets naturally cleaved off when the nucleotide(s) gets added by DNA polymerase; the DNA synthesized is also uninterrupted due to not having to stop, take a picture and remove the fluorophore manually. 2. this sequencer eliminates steric hinderance and noise.

Describe three differences between DNA and RNA.

1. The nitrogenous bases vary; for DNA the nitrogenous bases are Adenine, Thymine, Guanine and Cytosine whereas RNA nitrogenous bases are Adenine, Uracil, Guanine, and Cytosine 2. RNA has a hydroxyl group (-OH) on the 2' carbon of the 5-carbon sugar whereas DNA has a hydrogen (-H) (note: this is where the name *RIBOnucleic* acid and *DEOXYRIBOnucleic* acid comes from) 3. DNA is a double strand and has major and minor grooves whereas RNA is a single strand with only major grooves

In what two ways did the development of the TOPO cloning vector make cloning easier?

1. allows cloning to be a one-step procedure with no post-PCR procedures 2. allows cloning to be performed without ligase

Describe some of the ways that E.coli K12 has been modified to increase cloning efficiency.

1. restriction-modification is removed 2. DNA recombination systems are modified to prevent rearrangement 3. endonuclease activity is mutated to increase plasmid yield

Does A DNA or Z DNA have a right handed helix?

A DNA

Is A DNA or Z DNA the dehydrated form?

A DNA

When attempting to clone a specific cDNA, what are degenerate primers, AND why might they be advantageous?

A degenerate set of primers are primers that have some of its positions altered with different bases. This is useful because the primers would cover many different nucleotide combinations for proteins sequences. They are helpful for targeting protein sequences by covering all possible nucleotide combinations for that protein.

In QPCR, what does a lower Ct value represent in terms of relative RNA concentration in the starting sample? Why?

A lower CT value indicates that expression levels are high. This indicates that the relative RNA concentration in the starting sample was low. There is a higher amount of the target material, so it is able to be detected faster- resulting in a lower CT value.

What is a shuttle vector? What additional features (beyond #2) would you want in a shuttle vector? Why?

A shuttle vector can replicate in multiple host to avoid host incompatibility, you would want a autonomous replicating sequence to copy the plasmid in yeast, a selectable marker for yeast (URA3), and a yeast centromere to ensure segregation of the plasmid

What is a supershift and how does it enhance an EMSA experiment?

A supershift is when an antibody against the protein is added to the protein of interest. The antibody binding creates a greater molecular weight compared to just the DNA-protein complex, and thus causes a supershift where it is higher on the gel than both the bare DNA and the DNA-protein complex. The addition of the antibody creates another layer of specificity in the interaction of the protein with the DNA sequence.

In Maxam & Gilbert Sequencing: Why do you need an acrylamide gel?

Allows DNA to be separated by size by a single nucleotide

In an experiment designed to determine if Per1 protein levels are increased after treatment of cells with aldosterone, what would be an appropriate positive control? Why?

An appropriate positive control would be to probe for something that it is known will show a change. In this case, probing for Sgk was the positive control since it is known that treating Sgk with aldosterone increases levels of Sgk.

What is an inducible vector?

An inducible vector is one that has tight regulation of gene expression so that only certain environmental conditions will allow for the expression of genes/proteins in that system. Expression can be turned on via a specific substrate or enzyme being introduced to the environment.

In the analysis of Per1 protein expression: What is the primary antibody?

Anti-Per1 antibody, an antibody that is specific to Per1.

In addition to an appropriate vector, what else do you need to perform SDM?

Antibiotic selection marker, origin of replication, multiple cloning sites, synthetic oligonucleotides (primers)

What is the meaning of the term antiparallel in the structure of DNA?

Antiparallel in the structure of DNA refers to how each strand of the double helix is parallel to each other, but their orientation is opposite; their 5' - 3' polarities are in opposite directions.

In Sanger Sequencing: How does the automated approach reduce the number of reactions from 4 to 1?

Automated DNA sequencing utilizes primers labeled with a fluorescent dye instead of 32P, with each ddNTP type (ddGTP, ddATP, ddTTP, ddCTP) represented by a different fluorescent color. This allows for all four reactions to be mixed because the ddNTP that terminates the chain is represented by a specific color and therefore distinguishable.

How would I purify a protein if the protein has 'his-tags'?

Begin with bacteria cells that have been transformed with plasmids and have been induced to express protein of interested. Lyse the cells and run mixture through a Ni column. The Ni will bind to the his-tag of the protein and be bound to the column. Everything else will be washed away with buffer. Adding Histidine or Imidazole will obstruct the interaction and you will be able to collect the protein of interest. Addition of Enterokinase will cleave the his-tag at the end terminus. The sample can be run through column again, resulting in the purify protein of interest.

What is the main principle behind bisulfite PCR?

Bisulfite PCR is used to determine the methylation status of every C in a DNA sequence. CpG dinucleotides, a C nucleotide followed by a G nucleotide, are underrepresented in the mammalian genome, and tend to be clustered in regulatory regions such as a promoter region. Hence, this method is useful in understanding eukaryotic gene regulation. Methylation (hypermethylation) of CpG dinucleotides results in silencing gene expression. Treating a DNA sample with bisulfite results in all C's converted to U's. However, conversion doesn't happen if C is methylated.

What is the importance of blocking in a Southern Blot?

Blocking with nonspecific DNA or protein decreases non-specific binding of the labeled probe to the DNA that is fixed on the nylon membrane. Bovine serum albumin (BSA) will coat the membrane and will help in making sure that will only get specific interaction with probe and DNA of interest.

In a cloning experiment, how do you get rid of the bacteria that do not take up the plasmid?

By using plasmids that have selectable markers with antibiotic resistance (ampicillin resistance gene). Cells that take up the plasmid will not grow on the select antibiotic (ampicillin)

In DNA, C4 is bonded to...

C5

In Sanger Sequencing: How are the different bases detected in capillary electrophoresis as compared to normal gel electrophoresis?

Capillary electrophoresis involves injecting DNA fragments from a 96-well plate into capillaries, which then pass through a detection apparatus where a laser causes fluorescence excitation of each ddNTP (ddNTP is labeled with a different fluorescent color). Each band is recorded as it fluoresces a different color and plotted on a graph to determine the DNA sequence. Normal gel electrophoresis involves loading DNA fragments into four lanes (A, T, G, C) on a polyacrylamide gel and detecting bands using autoradiography. For all four lanes, the bands are read from the bottom to the top of the gel.

Describe the basic difference between cassette mutagenesis vs. Quikchange and Inverse PCR mutagenesis.

Cassette: Utilize restriction enzymes to remove wild-type sequencines and replace with mutations Quikchange and Inverse PCR: use PCR extension of oligonucleotides that contains desired mutation

What are the two functions of DNA in cells?

Cellular DNA contains two functions. One is the passing of genetic information to daughter cells. The other is the storage of information for the creation of mRNA by the process of transcription.

What is the central dogma? Give two examples of RNAs that are exceptions to the central dogma. Why are they exceptions?

Central dogma can be stated as a DNA → RNA → protein pathway. Some viral RNA is the exception to it. One RNA virus encodes RNA-dependent RNA polymerases (RdRPs) to replicate and transcript based on the RNA template. Another is also RNA viruses encode reverse transcriptase to synthesize DNA based on the information on the RNA template.

What are the advantages of ChIP-seq over ChIP?

ChIP is used to study about protein-DNA interactions. It allows us to determine what DNA a specific protein is bound to. ChIP only allows us to look at the DNA where a protein of interest is bound. We need to set up a PCR reaction and run gel electrophoresis after ChIP. Whereas, ChIP-Seq not only allows us to find out the DNA that the protein is bound to, but also to identify every location in the genome where the interactions between the DNA and the protein of interest occur without performing PCR reactions.

PCR positive control

Come in the kit, control plasmid and primer, can run the product on the gel to make sure the PCR worked

DpnI Digest positive control

Control the plasmid and primer, blue and white colonies screening will show the ratio of wildtype to mutant plasmid

Ligation positive control

Cut the plasmid with one restriction enzyme, should ligate back together

What is DAPA?

DAPA is DNA affinity purification Assay , a specific method that allows the identification and analysis of protein complex components.

In gel electrophoresis, why does DNA move towards the positive pole?

DNA has a negative charge and is attracted towards the positive pole of the gel electrophoresis.

How is DNA organized in an interphase nucleus?

DNA in an interphase nucleus is not as compacted as in a metaphase nucleus. In interphase nucleus, DNA needs to be more accessible for transcription. DNA is loose and not condensed.

Why does denaturing DNA on a blot allow for the detection of fragments based upon sequence?

DNA is first denatured dsDNA to ssDNA in the gel to increase affinity of transfer from gel to membrane. Having ssDNA in the membrane allows for probes to anneal to the ssDNA.

Explain the role of each of the histones in the formation of chromatin

DNA is wrapped around a nucleosome, which is composed of eight core histones (histone octamer). Each histone octamer is composed of a H3-H4 tetramer and two H2A-H2B dimers. Linker histone H1 interacts with the core histones to bring them closer together in an up-down structure to form a tighter and more organized chromatin that is compacted 7-fold.

In Southern blot analysis: What are the three ways in which you can increase the stringency of a hybridization?

Decrease salt concentration (ionic strength) Increase hybridization and wash temperatures Increase duration of wash (washing conditions optimized)

Southern Blot Fragment Detection Method

Detection of radioactively labeled probes using autoradiography. Nylon membrane is exposed to a piece of film to detect presence of radioisotopes such as 32P.

What is EMSA?

EMSA stands for Electromobility Shift Assay and is a way of studying DNA-protein interactions using the principle that a DNA-protein complex will be larger and have a higher molecular weight compared to bare DNA, which can be observed when comparing them via gel electrophoresis. The radioactively labeled bare DNA (oligonucleotide primers) will travel further down the gel than the DNA-protein complex.