Which of the following sets of primers could be used to PCRa

Which of the following set(s) of primers could be used to PCR-amplify the target DNA sequence shown inbetween the dots? (Note: only one strand of the double stranded DNA template is shown below). For each set of primers, explain why they would or would not amplify the target DNA. 5\'–GGCTAAGATCTGAATTTTC...CGAGGAGATATCCGCTAAT......TGGGCAATAATGTAGCGCCTT-3\'

a) 5’-GGAAAATTCAGATCTTAG-3’; 5’-TGGGCAATAATGTAGCG-3’

b) 5’-GCTAAGATCTGAATTTTC-3’; 5’-CGCTACATTATTGCCCA-3’

c) 5’-CGGAAATTCAGATCTTAG-3’; 5’-CGCTACATTATTGCCCA-3’

d) 5’-GCTAAGATCTGAATTTTC-3’; 5’-TGGGCAATAATGTAGCG-3’

Solution

Ans) Among all sets of options Ans. b) is the correct answer.

b) 5’-GCTAAGATCTGAATTTTC-3’; 5’-CGCTACATTATTGCCCA-3’

As we very well know about the PCR and it\'s working methodology, Primer Designing is an important phenomenon for PCR.Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest.

Ans. b) is correct because with respect to target DNA sequence one that is complementary to a portion of the 5\'-3\' strand, and another that is complementary to the portion of the 3\'-5\' strand of desired DNA. These primers hybridize to the separated template strands and serve as starting points for DNA amplification. The enzyme, called Taq DNA polymerase, extends the annealed primers\' sequence by \"reading\" the strand and synthesizing the complementary sequence.

Set a) It is not complimentary according to both positions of 5\'-3\' and 3\'-5\'. Thus no PCR product is there.

Set c) It is also complementary 5’-CGCTACATTATTGCCCA-3’ for 3\'-5\' but not for other 5\'-3\'. Thus no PCR product is there.

Set d) It is complementary 5’-GCTAAGATCTGAATTTTC-3’ for 5\'-3\' but not for other 3\'-5\'. Thus no PCR product is there.

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