Degenerate PCR - Sequencing?

Hank Seifert h-seifert at nwu.edu
Wed Mar 9 11:38:33 EST 1994


In article <GERLACHJ.3.2D7B7997 at QUCDN.QueensU.CA> GERLACHJ at QUCDN.QueensU.CA (Jim Gerlach) writes:
>We have been doing PCR with degenerate primers (17mers, usually 256 fold)
>and obtaining products in the 150-300 bp size. After doing restriction
>digests to establish that we had produced some non-human products we are now
>ready to sequence. We could, of course, clone and sequence in a conventional
>fashion but I'm wondering what are the chances of doing sequencing using the
>degenerate primers. Anyone tried this (or some other nice trick)? Thanx for
>any sugguestions or help.
>
>Cheers,
>Jim
>-------
>Jim Gerlach                               Cancer Research Laboratories
>gerlachj at qucdn (BITNET)                   Queen's University
>gerlachj at qucdn.queensu.ca (InterNet)      Room A309, Botterell Hall
>tel (613) 545-6446                        Kingston, Ontario
>fax (613) 545-6830                        Canada K7L 3N6

Jim:
We had reasonable results using degenerate primers on amplified 
products using 32P-end-labeled primers in a cycle sequencing 
kit (fmole by Promega).  The sequences were light but readable 
and told us that we were not amplifying the gene we were 
looking for.  Unfortunetely, we tried this after we had 
screened about 20 clones by sequencing!  I don't remember the 
details but I think we lowered the annealing temperture during 
the cycling and used a higher amount of primer.
I hope this helps.
Hank Seifert
Northwestern University



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