Posted 19 August 2009 - 06:49 PM
I am new into genetics and sequencing. I have two doubts,will be grateful if someone could give me any help.
1. Could we use genomic DNA (isolated DNA) as template for sequencing PCR without doing the preliminary PCR step and eluting?
2. To study a single base pair change (monoallelic mutation) could we use sequencing ? Because if their is a point mutation in one chromosome and not in other,pcr will give both bands, we elute ,sequence, could we see this difference? Iam sorry , very difficult to express this doubt .
But hope someone will help me
Posted 20 August 2009 - 08:41 PM
I don't think that you can use genomic DNA directly for sequencing. The concentration of your region of interest will be too low for direct sequencing. Better make a PCR first.
I guess that you could also not do direct sequencing from the PCR product. You will get both reads of your mutation. I would recommend cloning your PCR product with TA vector. Every colony you pick will have only one sequence from one allele.
Posted 21 August 2009 - 05:44 PM
Posted 22 August 2009 - 06:26 AM
Thank you very much for the answers,