Question

1. a. Why is it important to use a sequence region of high dissimilarity to make a strain-specific primer?

b. Why does only one primer, and not both, need to target a highly dissimilar region?

Answer 1

a) Primers are oligonucleotide sequences that bind to their complementary sequences. This allows the Polymerase to sit on the primers and initiate the process of polymerization. If the primers are designed for sequence regions that are not dissimilar, then the primer can bind to the sequence in any of the similar strains. As a result, upon amplification, the sequence can come from any of the strains. However, if it is designed for a dissimilar sequence region, then the amplified product becomes strain-specific. This is because the dissimilar region will not be present in other strains and as a result, the sequence will not be amplified from the other strains.

b) The PCR works by the addition of two primers namely, the forward and the reverse primer. The forward primer copies one strand while the reverse primer copies the complementary DNA strand, thereby leading to the amplification of the target sequence. Now, to amplify the sequence in a strain-specific manner, the primers have to be designed for dissimilar sequences. Hence, both forward and reverse primers can be designed to bind to dissimilar sequences to get the desired product. However, this would lead to a different primer pair for each of the strains, thereby increasing the cost and decreasing fidelity. Instead, only one of the primers can be designed in a strain-specific manner for the dissimilar sequence. The other primer can amplify one of the strands from each strain but will not be able to amplify the entire sequence as such as the complementary primer will fail to recognize this newly amplified segment from the other strains. Hence, at the end of PCR cycles, only a specific sequence will be amplified. This allows the design of only one primer in a strain-specific manner while keeping the other primer constant, thereby reducing cost and increasing fidelity.