Nucleic Acids Research, Vol 27, Issue 8 1819-1827, Copyright © 1999 by Oxford University Press
JP Day, RP Hammer, D Bergstrom and F Barany
A high sensitivity method for detecting low level mutations is under
development. A PCR reaction is performed in which a restriction site is
introduced in wild-type DNA by alteration of specific bases. Digestion of
wild-type DNA by the cognate restriction endonuclease (RE) enriches for
products with mutations within the recognition site. After reamplification,
mutations are identified by a ligation detection reaction (LDR). This
PCR/RE/LDR assay was initially used to detect PCR error in known wild-type
samples. PCR error was measured in low |Deltap K a| buffers containing
tricine, EPPS and citrate, as well as otherwise identical buffers
containing Tris. PCR conditions were optimized to minimize PCR error using
perfect match primers at the Msp I site in the p53 tumor suppressor gene at
codon 248. However, since mutations do not always occur within pre-existing
restriction sites, a generalized PCR/RE/LDR method requires the
introduction of a new restriction site. In principle, PCR with mismatch
primers can alter specific bases in a sequence and generate a new
restriction site. However, extension from 3' mismatch primers may generate
misextension products. We tested conversion of the Msp I (CCGG) site to a
Taq I site (TCGA). Conversion was unsuccessful using a natural base T
mismatch primer set. Conversion was successful when modified primers
containing the 6 H,8 H -3, 4- dihydropyrimido[4,5- c ][1,2]oxazine-7-one
(Q6) base at 3'-ends were used in three cycles of preconversion PCR prior
to conversion PCR using the 3' natural base T primers. The ability of the
pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to
greatly facilitate the conversion.
ARTICLES
Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations
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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