Question

Question: You determine that a particular gene in a peson with a genetic disease is the same size as that of a healthy individual. You suspect that the arises from a point mutation (a single nucleotide change that results in a defect in the protein.) Describe a how you could confirm this hypothesis.

Comment: We learned about Sanger Sequencing and Next Gen sequencing in class. Could you use either of these and if yes why would you use one over the other.

Answer 1

Point mutations that occur in non-coding sequences are usually without any results, although there are exceptions. If the mutated base pair is in the promoter sequence of a gene, then the expression of the gene could change. If the base change has occurred in the splicing site of an intron, then this could interfere with correct splicing of the transcribed pre-mRNA, and as a result the protein would also change. This is because, a change in one amino acid, could change the entire protein. Point mutations which occur in the genes related to metabolism could lead to beneficial as well as harmful traits or diseases.

For sequencing this particular gene, we could design a forward and reverse primer near this total gene sequence, amplify this particular sequence by Poymerase Chain Reaction (PCR) and then we could determine the base sequence of this particular region. For, sequencing purpose we could use the classical chain termination method from Sangers sequencing. After determing the sequence we have to compare with the specific sequence of the normal individual and look for point mutation. If there is any point mutation we could establish the hypothesis.