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).