PCR Direct Sequencing

tay-4 at bones.biochem.ualberta.ca tay-4 at bones.biochem.ualberta.ca
Sat Jul 16 15:23:34 EST 1994


In article <1188 at biosys.apldbio.COM>, inherit at apldbio.com (Inherit Account)
wrote:
> I have a question about PCR Direct Sequencing. Do you think what is most
>important thing to do that? 
> In PCR stage, the product is amplified well with no extra band, but sometimes
>I couldn't get good sequence result. Noisy. What is wrong?
> 
> And I try TA cloning but it need more time. I hope, it is possible, do PCR
>Direct Sequencing for every templates.
 
We had a graduate student in our lab a couple of years ago that used to 
perform a normal PCR reaction using a chromosomal template and then asymmetric 
PCR by just adding one of the primers in excess for a second round of 
amplification without any purification. Then he would purify the single 
stranded product from a gel and directly sequence that. It seemed to work fine 
for him, but then again, so did everything. This would eliminate the problem 
of the 2 strands reannealing.
I notice that Pharmacia has a new method that utilizes preferential 
phosphorylation of one primer and lambda exonuclease. After amplification, the 
phosphorylated strands are degraded by lambda exonuclease leaving only 
non-phosphorylated strands for single stranded sequencing. I wonder if that 
works?

Andrew



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