Question

Can you explain the answers below ?

Below is the same gene from page 1. Alternmative splicing of the primary RNA transcript will include either exon 4 or exon 5. Assume that mutants 1 and 5 are deletion mutants; mutant 2 is a frameshift mutation; and mutant 3, 4, and 6 are substitution mutations. PCR primers are designated with arrows and lowercase letters (a, b, c, d). mutant 4 mutant 61mutant 3 KR Kpnl Kpn ATG TAG TAA exon 1 exon 2 exon 3 exon 5 exon 6 exon 4 mutant 5mutant 2 mutant 1 Draw the expected bands if a PCR was performed with primers c and d. The arrowheads indicate the 3' ends of the primer. (3 points) c. WT 1

Answer 1

In this specific situation, we will not count for the alternative splicing because we are doing PCR which is a in vitro process where splicing enzymes are not provided.

For mutant 1 and 5, both are deletion mutation. So, some part of the nucleotide chain have been deleted from the wild type sequence. So, the PCR product will be shorter in length than the wild type. So, both are giving bands at lower molecular weight region than the WT.

As, mutant 2 is a frameshift mutation, the open reading frame have been changed. It is giving band to lower molecular weight than WT because after frameshift mutation possibly it is giving rise to truncated nucleotide chain. In other word, as the frame have been shifted, possibly the binding site for d primer have shifted to earlier sequences and giving shorter molecular weight nucleotide chain than WT.

Mutant 3,4 & 6 are substitution mutations, that means only one base change is present in the reading frame which we cannot distinguish visibly by the band. So, the bands will look similar to the WT band.