Cj. Fuery et al., Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: Assay design and optimization, CLIN CHEM, 46(5), 2000, pp. 620-624
Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows
detection of point mutations, deletions, and insertions. Reactions require
concurrent activity of a restriction endonuclease (RE) and a DNA polymerase
, both of which must be sufficiently thermostable to retain activity during
thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification
of sequences containing the RE recognition site, thus producing selective a
mplification of sequences that lack the RE site.
Methods: Assays were used that allowed the selection of conditions that pro
duce concurrent RE/DNA polymerase activity. The RE thermostability assay in
volved thermocycling a RE under various conditions and assessing residual c
leavage activity at various time points. Conditions found to preserve RE ac
tivity during thermocyling were then tested for their compatibility with DN
A polymerase-mediated PCR.
Results: A range of conditions that preserve activity of the RE BstNI over
30 cycles of PCR was identified. A subset of these conditions was subsequen
tly found to mediate specific amplification using Taq DNA polymerase. These
conditions were used to develop a REMS-PCR protocol for the detection of m
utations at codon 12 of the K-ras gene. This protocol allowed the detection
of 1 mutant allele in a background of 1000 wild-type alleles, The presence
of primer sets for RE and PCR control amplicons provided unambiguous asses
sment of mutant status,
Conclusion: Implementation of the assays described may facilitate developme
nt of REMS-PCR assays targeted to other loci associated with disease. (C) 2
000 American Association for Clinical Chemistry.