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overlap in the DNA sequence


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#1 Anoop Bhaskaran

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Posted 29 May 2013 - 04:49 AM

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?

#2 GNANA

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Posted 29 May 2013 - 05:03 AM

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.
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#3 Anoop Bhaskaran

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Posted 30 May 2013 - 12:46 AM

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..

#4 phage434

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Posted 30 May 2013 - 04:57 AM

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.

#5 Anoop Bhaskaran

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Posted 31 May 2013 - 03:36 AM

260 - 280 bp region ..mononucleotide A repeats. Overlap starts from 280 bp to end of the sequence. The expected length 600 bp

#6 phage434

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Posted 31 May 2013 - 03:55 AM

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.

#7 Anoop Bhaskaran

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Posted 01 June 2013 - 03:29 AM

I didn't try that..




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