Question

You utilized a transposon system for a forward genetic screen, creating random disruptions throughout the genome and screening for phenotypes. You found a mutant with a really interesting, never before seen phenotype.
How could you figure out what gene is affected?

Answer 1

Transposon insertion mutagenesis is a reverse genetic approach (Phenotype to genotype).
To determine which gene is affected that resulted in the given phenotype
1. Map the mutation using recombination frequencies
Strategy to map a recessive mutation using map-based cloning:
i. Cross the homozygous mutant strain with the WT strain and obtain F1
ii. Allow F1 to self (F1 female X F1male)
iii. Grow F2 individuals
iv. Select individuals with the mutant phenotype in the F2 generation (Mapping population)
v. Use different molecular markers to map the location of the mutation.
In the F2 mapping population, the individuals are homozygous at the mutant locus as they exhibit the mutant phenotype. However, the surrounding genomic regions do not necessarily come from a single parent due to crossing over and recombination during meiotic division.
As we move closer to the mutation, the recombination frequency decreases. Once we narrow down the mutation to a reasonable DNA boundary (50 kb - 150 kb), we can look for candidate genes. We can perform an allelic test with the known mutants in the region. We can also sequence the entire boundary region to identify the mutation.

2. If we know the sequence of the transposon, we can perform inverse-PCR and identify the adjacent sequence. It is a rapid process.