task One of your friends plans to make a series of polymerase chain reactions (P
ID: 274370 • Letter: T
Question
task
One of your friends plans to make a series of polymerase chain reactions (PCR). He has therefore purchased three sets of primers in the hope that one of the pairs of primers will be able to amplify the following template sequence:
The primer pairs look as follows:
1) None of the primer pairs shown give any product in a PCR reaction. Examine each primer pair and explain why they did not work. Indicate among other things if both primers are associated with errors or if only one primer in one pair poses a problem.
2) Your friend will not buy new primers. He therefore asks you for advice on how to save his attempt by combining two of the existing primers in a new way. What do you advise him about?
3) PCR is performed using a thermostable DNA polymerase. Why is this a big advantage?
Explanation / Answer
1) a good primer should have optimum length between 18 to 22 bp. Melting temperature in the range of 50 to 60 degree C. Absence of dimerization capability, absence of significant hair pin loop formation, lack of secondary priming structures.
Here, in both sense and antisense primer pair , length is too small. Along with that in primer pair 1) pair a has GC content more than 60% , highGC content meanshigh Tm which lead to secondary artefacts instead of PCR products. Along with that primer pair 1 have cross dimer intermolecular interaction at some bases which lead to the no product in PCR reaction.
In pair 2 there is also a interaction between both primer pair lead to no product.
5' GGACTTG 3'
3' CAG GTC G 5'
Primer pair 3 also undergo cross dimer formation at bp 4, 5 and 6 position because at that oint they are complementry of each other.
In primerpair 1 primer a is responsible where as in other pairs both primers are responsible for no product in PCR reaction.
3) thermostable DNA polymerase is used to run PCR at high temperature which facilitate high specificity of the primer and reduce the proportion of non specific product. So it remain stable at temperature variatiin in PCR and also eleminate need of add new enzyme to each round of thermocycling.
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