Nucleic Acids Research, Vol 27, Issue 8 1810-1818, Copyright © 1999 by Oxford University Press
JP Day, D Bergstrom, RP Hammer and F Barany
We compared the efficiency of PCR amplification using primers containing
either a nucleotide analog or a mismatch at the 3' base. To determine the
distribution of bases inserted opposite eight different analogs, 3' analog
primers were used to amplify four different templates. The products from
the reactions with the highest amplification efficiency were
sequenced.Analogs allowing efficient amplification followed by insertion of
a new base at that position are herein termed 'convertides'. The three
convertides with the highest amplification efficiency were used to convert
sequences containing C, T, G and A bases into products containing the
respective three remaining bases. Nine templates were used to generate
conversion products, as well as non-conversion control products with no
base change. We compared the ability of natural bases to convert specific
sites with and without a preconversion step using nucleotide analog
primers. Conversion products were identified by a ligation detection
reaction using primers specific for the converted sequence. We found that
conversions resulting in transitions were easier to accomplish than
transversions and that sequence context influences conversion.
Specifically, primer slippage appears to be an important mechanism for
producing artifacts via polymerase extension of a 3' base or analog
transiently base paired to neighboring bases of the template. Nucleotide
analogs could often reduce conversion artifacts and increase the yield of
the expected product. While new analogs are needed to reliably achieve
transversions, the current set have proven effective for creating
transition conversions.
ARTICLES
Nucleotide analogs facilitate base conversion with 3' mismatch primers
Department of Microbiology, Box 62, Hearst Microbiology Research Center, Strang Cancer Prevention Center, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
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