Inosine in PCR primers

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John Cooper writes:
>
>I was wondering... I've use inosine in degenerate primers and found that 
>they were read in sequencing gels as g's also. But I Interepreted the 
>primers as 'selecting' for G's. Do you have a reference for this or could 
>you elaborate more? We discarded the use of inosine for this reason when 
>making degenerate primers. You suggest our reasoning was faulty... Thanks in 
>advance.
>
>John Cooper
>NIH-NHLBI-LCM
>301-496-3150
>

John,
My reply to your address bounced so I hope that you don't mind me posting
this to methods. If Inosine only paired with C s then it wouldn't be useful
as a wildcard base for reducing degeneracy, would it ?. I don't have a
reference
for the observation that I residues in oligos, after cloning show up as G in
sequencing but all of my data indicates that this happens. I can offer two
possible reasons why this is.

1) When Taq polymerase (or Ecoli DNA pol) comes upon an I, it reads it as a G
and inserts a C opposite it.

2) When DNA containing I is propagated in E coli the Is get repaired to GC
pairs, regardless of what the I was paired with.

I think that 1) is more likely to be what's really happening.

Hope this helps

BILL



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William D. WARREN, PhD
Center for Agricultural Biotechnology     Email: ww40 at umail.umd.edu
University of Maryland at College Park    Phone: (301) 405-7681 

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