Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Sequencing shows different results when sequenced from the other end - (Jan/03/2012 )

I have a problem.

My sequencing chromatograms look fine and I have sequenced human PCR fragents to detect certain SNP's.

Now some SNP's are picked up by Sequecncing with the forward primer and are very clear heterozygotes.

When I then sequence from the other end, the apparent heterozygotes are very clear Wildtypes.

So Heterozygote (A/G) when sequenced from forward strand and Wildtype (A/A) when sequenced from reverse strand.

This is repeatable as well. Every new Product generated and sequenced shows Heterozygote from the front and Wildtype from the back.

PCR is done with Standard Taq Polymerase, the PCR Product is column purified and then sequenced (no cloning).

Any ideas what is wrong and what is the actual result? Het? WT?

Thanks a lot.


Get a proof reading polymerase - taq is well known to have difficulties with some bases, especially if they are repetitive.


I thought of that as well, but what I am seeing is no replication slippage but one specific base being heterozygote when sequenced from one end and Homozygote from the other end. No repetitions included


(assuming sanger sequencing) how far from the primer is the snp? if it is too close or too far then the result may be noisy. you have to look at the electropherogram to see if the base is there but not called (or if it is miscalled).