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

摘要: Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity a restriction endonuclease (RE) DNA polymerase, both which must be sufficiently thermostable to retain during thermocycling. The inclusion the RE in REMS-PCR inhibits amplification sequences containing recognition site, thus producing that lack site. Methods: Assays were used allowed selection conditions produce RE/DNA polymerase activity. thermostability assay involved thermocycling under various assessing residual cleavage at time points. Conditions found preserve thermocyling then tested for their compatibility with polymerase-mediated PCR. Results: A range Bst NI over 30 cycles PCR was identified. subset these subsequently mediate specific using Taq polymerase. These develop protocol mutations codon 12 K- ras gene. This 1 mutant allele background 1000 wild-type alleles. presence primer sets control amplicons provided unambiguous assessment status. Conclusion: Implementation assays described may facilitate development targeted other loci associated disease.