Mutation Screening - DNA Sequences... - (Aug/07/2007 )

Hi Friends,
Iam screening for mutations in the some of the genes of Vibrio cholerae, by sequencing the PCR products... My sequences have 'N's inbetween (non specific)... How should I consider this (as mutations???), coz recently when I went thru a mutation screening study, the authors have considered the 'N's as mutations (Heterozygous conditions)... Kindly clarify...

Did you call the bases yourself or did you let the computer do it? I don't know what they're talking about- you'll get many N's when the big-dye didn't work very well, or the capillery is dirty, etc.

I don't work with heterozygous screening but can't you clone your pcr product into bluescript or something. Than transform, and if you pick just one colony, you'll have a single sequence- any N's would be a consequence of technical issues in the sequencing.

If its 'N' s then you need to check the actual spectra to verify it. And you still cannot figure out with the raw data, then you need to resequence it again to make sure there is no mutation in the region where there are N's. if there is a mutation, then its has to be a base other than the actual base, which should be A,T, G or C and not N.

repeat the sequencing and it should be fine.