Question

1. Assume that another allele of the cinnabar gene, named cn^x, has a 1.8 kb insertion in exon 3. Develop a PCR strategy that would discriminate between cn^x and cn⁺ (include a schematic!); you can assume that the sequence of the cn^x allele is known. Include the time you would allow Taq polymerase to synthesize the DNA.

Note: In your answer you do not have to consider the cn¹ allele at all!

Answer 1

If a 1.8kb insertion in exon 3 has occured in the cnx allele of the cinnabar gene, then the PCR conditions to differentiate cnx from cn+ allele will be to change the extension timing only and to increase the final extension timing. Assuming that the length of the cnx allele is 500bp and the normal PCR extension temperature will be 72C for 30sec for a 500bp gene. But due to the insertion of the 1.8kb insert the length of the allele changes to 2.3kb. According to the standard PCR protocols, for every 1kb rise in the gene length 1min should be increased and hence the extension time should be increased to 2min and 30sec. at 72C. All the other temperatures and times will be normal as the normal PCR conditions. The number of cycles for both the alleles would be 25 because if the cycle number increases then other variables should also be increased.

Hence the same primers can be used for both the alleles. PCR condiitions will be:

1. Denaturation: 95C for 2min

2. (i) Denaturation 95 for 30sec

(ii) Annealing 55C for 15sec

   (iii) Extension 72C for 2min 30sec

Repeat step 2 for 25 cycles

3. Final extension 72C for 10min

4. 4C for infinity

The allele that has the insert will be a 2.3kb band on gel and which doesnt have the insert will be a 500bp band only.

If you have any query kindly comment before giving thumbs up. Thank you.