Jp. Day et al., Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations, NUCL ACID R, 27(8), 1999, pp. 1819-1827
A high sensitivity method for detecting low level mutations is under develo
pment. A PCR reaction is performed in which a restriction site is introduce
d 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 ar
e 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 erro
r was measured in low \Delta pK(a)\ buffers containing tricine, EPPS and ci
trate, as well as otherwise identical buffers containing Tris. PCR conditio
ns were optimized to minimize PCR error using perfect match primers at the
MspI site in the p53 tumor suppressor gene at codon 248, However, since mut
ations do not always occur within pre-existing restriction sites, a general
ized PCR/RE/LDR method requires the introduction of a new restriction site,
In principle, PCR with mismatch primers can alter specific bases in a sequ
ence and generate a new restriction site. However, extension from 3' mismat
ch primers may generate misextension products. We tested conversion of the
MspI (CCGG) site to a Taqi site (TCGA). Conversion was unsuccessful using a
natural base T mismatch primer set. Conversion was successful when modifie
d primers containing the 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazine-7-one
(Q(6)) base at 3'-ends were used in three cycles of preconversion PCR prio
r to conversion PCR using the 3' natural base T primers. The ability of the
pyrimidine analog Q(6) to access both a T-like and C-like tautomer appears
to greatly facilitate the conversion.