how would you detect a point mutation in the genome? PCR? - (Feb/28/2007 )

Have you ever come across the problem to detect a single nucleotide change in the genome?

Would you design a PCR primer for this task? If so what kind of primer, the mutated base at the 3'? If the PCR still gives you a product for both mutated and WT genome, what then?

If the mutation produces a restriction site you can use that of course; what if it doesn't?

Best, j

Why not just sequence the area of interest?

Yep, that would work if you only have a few samples. But if you have dozens of mice to genotype it's cumbersome.

Thanks, j

If you have DNA from dozens of mice, why not pool an aliquot from each mouse, do a PCR for your region of interest on this pooled DNA, and sequence the PCR product. If your product is clean (you may need to cut out a band from an agarose gel) so the sequence is of good quality, you can identify SNPs by comparing the chromatogram to a control chromatogram (one from a PCR product of a single homozygous mouse would be OK). The presence of a secondary peak under the primary peak (along with a reduction in the height of the primary peak) indicates the presence of a SNP at that position, and the relative peak heights can give you an estimate of the allele frequencies in your population. This method can detect SNPs with minor allele frequencies down to about 5% or so.

Do you want to genotype individual mice? If so, there are lots of genotyping systems around based on either hybridisation or primer extension. Allele-specific PCR can work well if you can optimise your reactions to amplify just one allele preferentially, but this technique doesn't always give reliable, reproducible results. Besides, the absence of a PCR product is not a good way to show the absence of a specific allele - too many other ways of getting no PCR product, as we all know.