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Old 05-31-2012, 03:35 AM   #1
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Location: UK

Join Date: Mar 2012
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Default Sequencing advice

I am planning to sequence 60kb gene using ovelapping long range PCR, I have designed primers and amplified whole gene. I am running into problem now because I am amplifying mixtures of sequences of another gene that is highly homologous (93%) to my gene.

When I sequence mixture of amplified population, will I will be able to characetrize SNPs specific to my gene by aligning to reference genome?
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Old 05-31-2012, 11:33 AM   #2
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If it is so homologus, you are bound to see mis-alignment. It might be a good idea to check out mappability features for your genes of interest. Perhaps a longer read application would be more suited for you..
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Old 05-31-2012, 01:23 PM   #3
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Always BLAT your primer sequences prior to having them synthesized. If they map to more than a single region in the genome redesign them.
NextGenSeq is offline   Reply With Quote

alignment, pcr sequencing

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