Detection of Rare Mutant Alleles by Restriction Endonuclease-mediated Selective-PCR: Assay Design and Optimization

BACKGROUNDRestriction 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 amplification of sequences that lack the RE site.METHODSAssays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR.RESULTSA range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations 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 assessment of mutant status.CONCLUSIONImplementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.