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.
Department of Genetics
University of Dublin e-mail: khwolfe at tcd.ie
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