Sequence variations between strains

esylander at NETMAIL.HSCBKLYN.EDU esylander at NETMAIL.HSCBKLYN.EDU
Wed Jan 22 14:09:45 EST 1997


The best polymerase for good fidelity is from NEB. We have used the Vent
polymerases to good effect.

Beth Sylander
SUNY Brooklyn
_______________________________________________________________________________
Subject: Re: Sequence variations between strains
From:    khwolfe at tcd.ie (Ken Wolfe) at Internet
Date:    1/22/97  1:46 PM

In article <Pine.SOL.3.95.970121131933.5665A-100000 at terre>,
webers at IRCM.UMontreal.CA (RAY-Sandra Weber) wrote:

> We are currently PCR cloning a DNA fragment from the yeast genome using
> PFU polymerase and have noticed differences (eg., 6 discrepancies per 200
> base pairs) between the sequences generated from the Genome Sequencing
> Project and our data.  Our template DNA is derived from W303-1A which is
> different from the strain used in the Genome Project.  My questions are a)
> Has anyone else observed strain-dependent sequence variations or can they
> be attributed to PFU polymerase error? b)  If these errors are due to PFU,
> what conditions or other polymerases could be used to increase fidelity?
> (We typically use between 0-0.75 mM Mg++, 200 uM dNTPs, 2.5-5U of PFU and
> 30 cycles of amplification).

This sounds like a polymerase problem.  There are 21 yeast sequences in
GenBank derived from strain W303, totalling 59 kilobases.  Comparing these
to the genome sequence (strain S288C) with the BLAST program reveals 99.4%
sequence identity but 50 gaps, i.e. 1 gap per 1200 bases.  Most of the
gaps are single nucleotide insertions/deletions in noncoding regions.  One
gene (SDC25) is a pseudogene in S288C but alive and well in W303.

Ken

-- 
Ken Wolfe
Department of Genetics
University of Dublin                    e-mail: khwolfe at tcd.ie
Trinity College                         phone:  +353-1-608-1253
Dublin 2, Ireland                       FAX:    +353-1-679-8558


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