Question

The normal (wildtype) size of the brat gene is 1500 base pairs (bp). In the mutant variant you identified, there is a deletion of 500 bps in the last exon of the gene. How would you recognize the two versions based on size? List what fragment sizes you would expect to see on a gel.

Answer 1

The identification of the two variants can be done with PCR using two different primer sets. As there is a deletion in the last exon of the gene, primers designed for the amplification of the full-length brat gene will not succeed in the amplification of mutant variant. So another primer that binds to the sequence before the deleted fragment will be used. Below figure shows the strategy for designing the experiment

Two PCR amplification will be done in each sample- wild type brat and mutant brat using promer set P1/P2 and P1/P3. the resultant PCR amplification after gel run will be as below.

In the wildtype sample, there will be amplification in both the primer combination. However in the mutant, because of the deletion of 500bp, the primer combination P1/P3 will not lead to any amplification in the PCR and only the PCR combination P1/P2 will lead to amplification with amplicon size of 1000bp.