Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: Assay design and optimization

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.