N-1 sequencing problem

[Stewart Payne]

I run a clinical molecular genetics diagnostic lab. Approximately 70% of the output is sequencing disease genes for pathological mutations. In recent weeks we have been hit by major problems where sequence quality is trashed by what appears to be n-1 products in the sequence traces. All primers (for all amplicons across many genes) are M13 tagged to allow universal sequencing of a large variety of templates. The n-1 problem goes across the board. We have had new M13 sequencing primers synthesised to the highest purity and experiments suggest the problem is template-related. I cannot understand why N-1 template should give N-1 sequencing product from a pure sequencing primer. Any suggestions??

[Daniel]

I suspect that you may have PCR contamination of your primers or PCR buffer. This could then give rise to PCR product in the sequencing reaction. You may want to try doing a sequencing reaction without any product and see what you get.

Daniel Tillett
Nucleics Suppo

[Author]

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