Virtual Experiment 7 based on practicals PDF

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15.5/20. Tough questions which change every year however this can be used as a reference....

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Virtual Experiment 7 – Molecular Biology 1 1. Thermostable DNA polymerases exhibit activity profiles such as the one shown below for a Taq polymerase. The PCR tubes for Experiment 7 are, for convenience, set up at room temperature (RT) until being loaded into the thermal cycler for the amplification process, in which the protocol specifies that each extension step in the cycles be carried out at 72C for 2.5 min. In one of the PCR cyclers, the technician accidently set the extension temperature at 65C. Would the product yield be the same as that from the 72C extended product? Explain your answer briefly. [2 marks] [Clue: Answer is in the graph below and extension time.]

In short, no, the product yield would not be the same. As can be seen in the diagram above, the 72℃ extension temperature is optimised to attain the highest extension rate and greatest yield. Thus, if the extension temperature was set to 65 ℃, the resulting product volume would be significantly less due to the reduced extension and optimisation rate.

2. Two honours students have related projects that use the lmrA gene. Their supervisor asks them to begin their projects by producing a large amount of lmrA DNA, using PCR and the same bookended primers as used in this experiment. In a slightly confusing situation, one student uses the pAMG25 plasmid as the template, but the second student finds a small amount of the actual lmrA linear double-stranded DNA. Both proceed independently with their PCRs. Has one of the students acted incorrectly? What do you expect the two PCR outcomes to be when the students view their products in an agarose gel? [2 marks] [Clue: Check the source of the primers.] It is possible to successfully perform both PCR reactions with the primers sourced from different origins. Two slightly different processes will be used, and the result will look different for both methods; however, successful extension is possible. On the resulting gel electrophoresis result, the bands from the plasmid derived primers will be closer to the origin above the original templates in the gel. In the linear derived primer scenario, the gel bands will arrange in line with the original template strands or further away from the origin.

3. You are planning a PCR experiment for which you have designed forward and reverse primers as shown here: Forward: 5'-ATGGCTTAAAGCTACATGAAAGAGGTCCACAAATGGCCAAT-3' Reverse: 5'-TTGGAAGGCTAAGAATTGACCAACAGTCAATTGTTCTGAAAC-3'

You now need to calculate the Tms of these primers to see if they are suitable for your PCR. A useful online tool for calculating primer Tms is the Promega Biomath Oligo site: https://www.promega.com.au/resources/tools/biomath/tm-calculator/ When you enter the site, all you need to do is enter the forward oligo sequence in Step 1 and choose the first GoTaq Green buffer system in Step 3 and hit the Calculate button. Repeat for the reverse primer. From the calculated Tms, are your primers suitable or unsuitable for your PCR? Explain. [3 marks] [Clue: Primer Tms calculated from the Biomath site should enable you to answer this question.] Forward Tm: 75℃ Reverse Tm: 73℃ The melting temperatures for these two primers make them suitable as the respective temperatures are close together. Thus, annealing of the two strands during the PCR reaction will be more efficient compared to two primers with a greater difference between melting temperatures.

4. This question concerns the gel photo on the Result Sheet. The PCR results in lanes 3-6, where the annealing temperatures were 50 ℃-65 ℃, seem to have worked equally well. (a) What would you expect if we had used an annealing temperature of say 40℃? (b) Can you explain the results in lanes 7 and 8, given that Taq polymerase is still very active at these higher temperatures? [3 marks] [Clue: (a) Too low an annealing temperature will cause the primers to make mismatched bindings on the DNA template. (b) All about primer Tms.] a) If an annealing temperature of 40 ℃ were utilised, a significantly reduced product yield would be obtained. This is because decreasing temperatures increase the mismatch binding of primers. Thus, it eventually gets to a point where the sequence has been altered in such a way that primers can no longer bind and extend. b) Lanes 7 and 8 anneal at temperatures of 70 ℃ and 75 ℃, respectively. The primers used, however, have melting points around these temperatures. Thus, little to no yield has been produced as the primers have not been able to bind during the annealing stage as a result of being denatured by the higher temperatures.

5. Calculate the theoretical product yield at the end of 15 cycles, which is halfway to the 30 cycles used in this PCR experiment. [1.5 marks] [Clue: A calculator will do this for you.] Theoretical Yield = 27 x 215 = 884736 nanograms x 10-9 = 0.000884736 grams

6. In this experiment, the extension time was 2.5 mins, comfortably long enough to copy the entire lmrA sequence, at ~1.7 kb, bookended by the primers, give the rule-of-thumb of ~1 kb/min. A student who was sceptical of this PCR extension rate wanted to do the same PCR but by doubling the extension time to 5 mins. The amplified product yields for the two different PCRs were examined in an agarose gel. What differences, if any, would you expect to see in the gel lanes? Explain your answer. [2 marks] [Clue: Shouldn’t need a clue, as the answer is more or less in the question.]

Increasing the extension time will ultimately result in increased volumes of non-bookended longer strands alongside increasing bookended strands. As a result, when gel electrophoresis is run the large volumes of longer strands will results in a smeared gel band.

7. You are planning a PCR experiment, and you are supplied with the set of primers shown below. What is wrong with this primer set? [1.5 marks] 5’-AACTGGATACACTAGCGACATGCCCAGAGACATACTCATGCGG-3’ 5’-GAATCTACACACTGGGGGGGGGATCCCCCCCCCCTAAACTGCC-3’ [Clue: Check the primer sequences.] Firstly, both primer sets are 43 base pairs in length each. The ideal length is between 18-24 base pairs, as anything more than that makes it difficult for the primers to bind to a perfectly complementary sequence. Additionally, the first primer does not begin with a G or C, and there are no restriction site palindromes for either primer sequence.

8. The picture below is a composite of separate sets of PCRs. Lane 1 is the template control. Lanes 2 to 5 and lanes 6 to 9 are from PCRs that used 1, 2, 5, and 10 ng of template, respectively, in otherwise identical reaction mixtures. You can just make out small amounts of product in lanes 4 and 5, but lanes 6 to 10 have copious amounts of amplified DNA. (a) Can you say which PCR parameter is different between these two sets of PCRs, that is, in lanes 2-5 versus 6-9? (b) Why are there seemingly equivalent amounts of amplified DNA in lanes 6 to 9, given that the starting template masses were so different? [3 marks] [Clue: (a) The left set is low yield, and the right set is high yield, yet both begin the PCRs with the same set of template masses. Only one parameter can cause the vast increase in yield seen in the second set of products (lanes 6-9)].

a) The PCR parameter that differs between these two gel results is the number of cycles run. Lanes 25 most likely have been run through 10 cycles of PCR which is not enough cycles to present significant masses on the gel. Lanes 6-9 subsequently have had 30 cycles of PCR, resulting in significantly increased masses and thus clear gel readings. b) Even though each band has a different starting concentration, the PCR reaction means that they all increase on an exponential scale. The length of the starting primer sequences is, therefore, the only factor that determines the final masses. As such, even though different starting concentrations were utilised, the sustained exponential increase of identical primers and primer lengths will result in similar final masses.

9. You are about to embark on a research project in which you begin by using PCR to amplify a gene of interest from a eukaryotic chromosomal DNA source. There are two types of thermostable DNA polymerases available in the laboratory, namely Phusion and Platinum Taq DNA polymerase and Pfu Ultra DNA polymerase. Check the graph below for the fidelity of thermostable DNA polymerases. Which one should you use and why? [2 marks] [Clue: Answer is in the graph.]

The Pfu Ultra DNA polymerase has a higher fidelity rate compared to the Phusion and Platinum Taq DNA polymerase relative to the original Taq polymerase. It should therefore be utilised in the extension phase of this PCR experiment as its checking and correcting enzyme as a lower rate of mispairing compared to the Platinum Taq. Thus, the PCR yield would be greatly increased with the Pfu Ultra DNA Polymerase....