problem in sequencing with one primer

[sandeep]

hi everybody there
I m getting problem with sequencing of my PCR product, the problem is that the sequencing when done with reverse primer comes out to be quite good but in case of forward primer the sequencing initiates well but after 50-60 bases multiple peaks each with very low intensity start appearing
I m quite confused where the problem lies
can anyone please help me to troubleshoot??

[FunctionalBio]

Hi,

I work for a sequencing service called Functional Biosciences, Inc so i have alot of experience with these types of problems.

If you’re doing some chromasomal DNA, its possible you have a heterozygous insertion/deletion mutation. If its an in/del you might be able to see a pattern where one of the chromasome’s sequence is shifted a few bases to the left or right. The two chromasome’s being different causes two sets of peaks, each half the height of a normal peak.

Does the forward primer reach all the way across to this specific point in the sequence? If the forward is able to go past this sequence without any changes, then its probably not an in/del.

[Anonymous]

Hi,

I’m working with DNA sequencing of Malassezia (yeast), analyzing ITS region and D1/D2 region 28S of ribosomal DNA. When I sequence the PCR products of those 2 regions (separately), using forward and reverse primers, I have problems only with the reverse primers. The quality of the bases is too low and noisy. The DNA sequencing with forward primers works good.

Could you help me with this problem?

Thanks.

ASN

[Author]

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