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Chapter 17: PCR, Quantitative PCR, and PCR Applications
(Exam 3) Questions with Accurate Answers
______ PCR allows for the amplification of a gene when only part of the sequence is known, and part is unknown or variable. Describe this method. Fun Fact: this method was used in genome walking, when researchers would start at one end of a chromosome, sequence the fragment, and use a primer from the fragment and an anchor primer to sequence the following segment of the chromosome. correct answer Anchored. A DNA polymerase (terminal deoxynucleotidyl transferase) adds a polyG tail to the 3' end of the DNA. An anchor primer with polyCs will bind to the polyG tail and from here the second strand will be synthesized. You can then amplify the dsDNA with regular PCR. ________ PCR uses the enzyme helicase to separate the two complementary DNA strands just as it occurs in the cell, and at lower temperatures than needed in traditional PCR. It's super quick, but require more template DNA and still needs to be optimized. It could allow for handheld PCR devices for use by researchers in the field or MDs. correct answer helicase-dependent (T/F) A plasmid can be used for site-directed mutagenesis in a single PCR. correct answer True. The plasmid is denatured and primers with the mutation will hybridize to this ssDNA and overlap with the region to be mutated. The primer/plasmid is copied with PCR, transofrmed into E. coli and ligated and copied. (T/F) PCR can be used to identify an organism's genotype. correct answer True. The method is called gene typing that uses PCR. (T/F) The reverse primer is identical to the Crick strand (complementary to the gene of interest) in the 5' to 3' direction. correct answer True. This could be generated by coming up with the complement to the gene of interest and reversing it. (T/F) When using site-directed mutagenesis to create deletions, the primer still includes the sequence of nucleotides that are to be deleted. correct answer False. The nucleotides to be deleted are completely left out of the primer so that the overhangs of the primers will interact during the final PCR.
(T/F)
PCR can be used to amplify ancient DNA. correct answer True. However, amplification is tough because of the following problems:
- low quantity -contamination -covalent modifications over the years. A ______is likened to a microarray to detect the levels of dozens of genes from a sample at the same time. correct answer qPCR array Another difference between traditional PCR and qPCR is the presence of Taq polymerase exonuclease domain in qPCR. Describe what it is needed for. correct answer Taq polymerase isn't needed in traditional PCR, but in qPCR it'll chew up any downstream DNA bound to the template (including the probe). The digestion (or cleavage) of the probe by polymerase is what causes the 5' and 3' ends of the probe to separate, which produces the color change by FRET that is observed. Describe asymmetric PCR. correct answer Assymetric PCR is a method used to generate ssDNA probes. An asymmetrical primer ratio is used 100:1, primer 1:primer 2. dsDNA is generated in 10-15 cycles until the supply of primer 2 is depleted, at which point ss DNA from the first primer (the non-limiting primer) is produced. This is good for DNA hybridization. Describe nested PCR. correct answer This PCR increases the specificity of PCR by reducing nonspecific product amplification. It uses four primers (first pair amps outside of the gene of the interest and second pair is nested inside of the first pair). The first PCR is for primers 1 and 2 (outside) and the second PCR is for the nested primers to amplify the gene of interest (1 and 2 were non-specific). This allows for amp of a very small amount of DNA in a sample. Describe RAPD PCR and some situations in which you would use it. correct answer RAPD PCR (Random Amplification of Polymorphic DNA) is used to compare similarities or differences between individuals. Short primers are used that will randomly bind to the DNA that act as sites for DNA polymerase synthesis. If there is a difference between two people (SNPS), the primer will bind differently and different DNA fragments will be produced. The fragments are analyzed by agarose gel. See page 204 for more information.
5' ATGCATCGT....ACTGGCATTTCGGAG 3' correct answer 5' CTCC 3' Remember that the reverse primer will be the reverse compliment How would you calculate the number of copies of DNA produced by PCR? correct answer 2^n number of cycles How would you generate a melt curve? correct answer Raise the temperature of the qPCR products slowly and measure the change in fluorescence as the DNA dissociates. If the DNA is pure there will be a single peak at 80-90 C range. Multiple peaks would mean multiple samples of DNA with different Tms. Peaks at lower temps would mean primer dimers or other small DNA contaminants with low Tms. If the reporter and the quencher are close, the quencher will absorb the energy from the reporter and the fluorescence from the quencher will be absorbed. Read what happens when the two are far apart. correct answer The probe will bind to the DNA at which point the Q will be able to absorb energy from the R (fluorescence from the Q will be detected). When DNA polymerase comes through it will chip off the Q and R, causing them to be far apart. When Q and R are separated, only the fluorescence from Q will be detected. Still having trouble? Watch https://www.youtube.com/watch?v=kvQWKcMdyS4 (you can skip ahead to get to the probe part) In lab practice, you would want primers that have pretty similar annealing temps (within a couple degrees of another). If you have two primers that aren't that close together, what could you do to alter the annealing temp to be more practical? correct answer Bases could be added or subtracted from either primer Interesting little fact. correct answer PCR variations have improved or replaced techniques we've talked about like RFLP, VNTR, and northern blotting Make sure to go over pages 194-198 and the info having to do with making a primer with cut sites, stop codons, tags (like a his-tag) etc. There was no simple way to ask questions about these. correct answer :) Outline the process of PCR. correct answer dsDNA template is denatured into ssDNA at a high temp, primer annealing to each piece of ssDNA, and extension (or elongation) of the primers by DNA polymerase at the 3' end, creating new dsDNA.
Outline which primers would be used for a 3-PCR Site directed mutagenesis. correct answer 1st PCR: External forward and internal reverse 2nd PCR: Internal Forward and external reverse 3rd PCR: External forward and external reverse. PCR can be used via ______ to create mutations, deletions, and insertions in a gene. correct answer Site directed mutagenesis. This technique was developed by Michael Smith who shared the Nobel Prize with Kary Mullis (who invented PCR). Site directed mutagenesis uses traditional PCR and various primers to produce alterations in a gene product. PCR can end up making unwanted products along with the gene if the primer binds non-specifically (binds to another site). These two variations of PCR are used to counter this by increasing PCR specificity. correct answer Touchdown PCR and nested PCR. PCR is limited to copying several thousand base pairs. This can be overcome in some cases by making Long PCR. Outline how you would accomplish this. correct answer You can make Long PCR by using Taq with Pfu and longer extension times. If this doesn't work, then REs and DNA ligase can be used to amplify shorter DNA fragments and join them together. qPCR data can be analyzed in order to compare samples to each other and determine the relative amount of starting material in the original samples. Describe how you would read one and what it means (what is the Ct and what does it mean if you see a curve cross the Ct earlier vs. later?) correct answer The Ct is a point at which you or the computer decided to compare samples. Curves will cross the line at the cycle number that the fluorescence is detected. A curve that crosses the line at an earlier cycle means that there is MORE DNA, which caused more cleaving of more probes to produce fluorescence earlier. A curve that crosses the line at a later cycle means that there is LESS DNA, less probes being cleaved, and therefore fluorescence is detected at a later cycle. Read this about solid state PCR. correct answer Solid state PCR performs PCR in oil (emulsion PCR) or on beads (bridge PCR). The product can be detected with 32P incorporation and restriction digestion. It's good because it reduces cross-contamination and doesn't require gel electrophoresis or cloning. It's high-throughput and can detect as low as 10K copies of template. It allows for the identification of SNPs, gene typing DNA sequencing, and is being used to detect cancers and pathogens from a single cell.
Start at ~2:10 for the probe portion and run until you've watched the detection portion. The (forward/reverse) primer is identical to the gene of interest. correct answer Forward The annealing temperature should be _______ than the lowest Tm of a pair of primers. correct answer 5 degrees less The DNA polymerase will read the template in this direction while the DNA will be synthesized in this other direction. correct answer 3' to 5' 5' to 3' The reagents of qPCR and traditional PCR are very similar. One thing that is present in qPCR but not in trad. PCR is a probe. Describe the role and function of the probe. correct answer The probe will bind to the template DNA somewhere between the forward and reverse primer. It takes advantage of FRET (fluorescent resonance energy transfer), which is basically a transfer between a quencher and a reporter that, once disrupted can be detected by a flurometer. (refer to video on other card) The temperature at which 50% of the primer is bound to the template DNA is known as the _____. correct answer Tm. Tm can be estimated by adding ___ degrees for every AT pair and ___ degrees for every GC pair. Why would the hand-calculated temperature not always be exact? correct answer 2, 4. Base stacking, base grouping, and hydrogen bonds must be taken into account. To generate the reverse primer you must generate the __________ from the Watson. (Hint: what is the relationship between the Watson/gene of interest strand and the reverse primer?) correct answer reverse complement What are some differences between traditional PCR and qPCR? correct answer traditional PCR: measures products once they've plateaued. Needs agarose gel electrophoresis to analyze the PCR product. qPCR: measures kinetics of PCR during logarithmic phase. No need for the agarose gel because you can quantify DNA at the same time. Presence of a probe (see other flashcard) and Taq polymerase.
What are some limitations of traditional PCR? correct answer -not quantitative (the end product is variable) and exact concentrations are unable to be determined without performing post-PCR processing. -Agarose gel electrophoresis is the standard technique for analyzing a PCR product, but it's time- consuming, subjective, and relies on EtBr binding consistently to the products for proper analysis. What are the two pairs of primers used in site directed mutagenesis? correct answer External forward and reverse and internal forward and reverse. What is an alternative to using a probe in qPCR? Explain how it works. correct answer Instead of using a probe, SYBR green dye can be used. This dye fluoresces when bound to dsDNA but not ssDNA. The more double stranded products being produced, the more SYBR green will be detected. What is ligation-independent cloning? correct answer Uses modified primer sequences that are comp. to a linearized plasmid. After PCR the amplified DNA and plasmid are mixed and primers will anneal to the plasmid overhangs, and in this case the insert directionality is sure. The product is transformed directly into E. coli which will ligate and copy the plasmid. What is multiplex PCR? correct answer Multiplex PCR is a method that uses more sets of primers to amplify multiple genes of interest. What is oligonucleotide stitching? correct answer This is a method where you can make long fragments of DNA by using overlapping sequences. Gaps in the fragment can be filled by DNA polymerase and sealed up with ligase. Researchers can thus make any sequence of DNA they need without having to do PCR and clone fragments to build the gene required. What is quantitative PCR (qPCR)? Note: This can also be called real time PCR or RT-PCR, but Borgon won't refer to qPCR as this because a reverse transcriptase PCR acronym also exists and it just gets confusing. correct answer qPCR is a way to take the variability out of traditional PCR by measuring the kinetics of the PCR reaction in its log phase. Traditional PCR will only measure the products once they have plateaued. It allows you to quantify and amplify DNA at the same time without needing agarose gel electrophoresis.
What would be used to account for the lack of specificity that SYBR green dye offers? correct answer melt curve What would happen if an annealing temperature at or above the Tm was used? correct answer The primer would likely not bind very well to the template DNA and no copying would occur. What would happen if an annealing temperature too far below the Tm was used? correct answer This would cause the primer to anneal nonspecifically to other sites on the template DNA, allowing polymerase to copy unwanted DNA. When you want to look at the regions flanking a gene in order to look at a gene's upstream regulatory elements that control its expression, introns, or intergenic DNA, you want to use _____ PCR. Describe this method. correct answer Inverse. A piece of DNA is digested with REs and circularized. It's digested again, which cuts the gene in half, flipping the DNA so that it's on the outside and the flanking regions are on the inside. Primers specific to the flanking region can now be used to amplify this segment. Who developed PCR? correct answer Kary Mullis. He showed that using a piece of template DNA and two primers could make billions of copies of DNA in a matter of hours. Why is PCR limited to only 30 cycles? correct answer The shelf life of TAQ polymerase is only 40 minutes and it can denature over time. Also, at a certain point there is too much DNA to copy and not enough enzyme to extend every single primer. Primers and dNTPS are limited and depleted at about 30 cycles.
