overlap in the DNA sequence - (May/29/2013 )
After sequencing the PCR proucts sometimes its get overlapped..the gel extracted PCR proucts send for sequncing. Did anyone face the same issue? pls help to know the reason behind the overlappin and how to overcome this problem?
In my view, overlaping peaks may be due to the problem with sequencing itself or it might even be genuine due to an heterozygous deletion. these you can predict when you look at your chromatogram; if the overlapin is right frm the start and the peaks doesnt look good then it mostly due to seq errors casued by impure/less sample or non-specific product or even bad sequencing. but when the overlapin starts in the mid suddenly, until then the single peak looked good, then it might be due to one of the allele has got a base/bases deleted and so the overlaping occurs with the successive base peaks. just think abt this and analyse ur sequence you 'll find an answer.
Thanks for the reply...the overlaps starts in the middle around 280 bp..as you said it is because of the alleles..i want to know more about it..pls help me. ..sequence is trnH - psbA from plant chloroplast genome. its a nocoding region and mononucleotide repeats are there..
How long is the repeat? You can sometimes determine the sequence of the next base following the repeat, which is often the same for both sequences. Then, a primer which is a poly A or poly T followed by that base can be used to sequence prime. You could also perhaps do an inverse PCR reaction and then sequence in the opposite direction.
260 - 280 bp region ..mononucleotide A repeats. Overlap starts from 280 bp to end of the sequence. The expected length 600 bp
If you sequence in the other direction, what does your sequence look like? Poly A regions often exhibit polymorphic behavior, either in from PCR or from genomic slipping.
I didn't try that..