Degenerate PCR - Sequencing?
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.
>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
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.
More information about the Methods