degenerate oligos: I or everything?
user at host.edu
Mon Dec 30 01:11:33 EST 1996
In article <Pine.PMDF.3.95.961228161453.1049B-100000 at OPAL.TUFTS.EDU>,
<jstrassw at OPAL.TUFTS.EDU> wrote:
> I would like to know what the preferrred oligo strategy is for reverse
> genetics. I have a peptide sequence and would like to PCR out the
> corresponding cDNA. So, I have two choices: either put inosine in the 3rd
> position or include all 4 bases at the redundant position. Any thoughts?
I have cloned a protein with primers that were 28-32 nt long with 5-6
inosines and 32-64 fold degeneracy. My experience shows that you should
try to put your degenerate sites in the 3' region of the primers and
inosines in the rest of the sequence. I try to keep the length of the
primers around 24 without inosines. It's been shown that you cannot have
inosines in the 4 3'most nucleotides or hybridization efficiency drops
significantly (I think I can dig out the reference). I also try to keep
the degeneracy down not to decrease the efficient oligo concentration. I
also found that all inosines were read as Gs, i.e. hybridized to Cs in
sequencing, just like the papers say (Yes, finally theory meets practice
;-) ), so they would probably do in PCR. And the melting T proves that
inosines are passive, i.e. do not add to Tm nor decrease it, so just
ignore them when calculating the Tm.
And there is also a PCR method with TMAC that is forming an additional
bond with A-T pair bringing its energy to that of G-C so degenerate pool
will have more close Tms. It is not as efficient as hybridization with
TMAC where you use 3M solution, when in PCR - 0.5-50 mM only before
inhibition. My experience with the method is limited.
Try touchdown PCR too - very powerful.
Good luck and may the Gods of PCR be with you (and patience)!
Albert Einstein College of Medicine.
oleyniko at aecom.yu.edu
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