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Sequencing doubts


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3 replies to this topic

#1 niveda

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Posted 19 August 2009 - 06:49 PM

Hi,
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 :lol: .
But hope someone will help me
Thankyou

#2 Rsm

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Posted 20 August 2009 - 08:41 PM

Hi,
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.
Cheers,
Minna
I got soul, but I'm not a soldier

#3 phage434

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Posted 21 August 2009 - 05:44 PM

I agree with Minna; for all except small bacterial genomes, direct sequencing of genomic DNA is difficult or impossible. You can PCR the region of interest, followed by sequencing. Often you can determine that a specific base has two different nucleotides, if that is the situation, but the cloning approach, along with statistical sampling of the clones, will provide greater certainty.

#4 niveda

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Posted 22 August 2009 - 06:26 AM

Hi,
Thank you very much for the answers,
thankyou again




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