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

¹ Johnson & Johnson Research Pty Limited, Australian Technology Park, Level 4, 1 Central Ave., Eveleigh NSW 1430, Australia

² Department of Medical Oncology, St. Vincent’s Hospital, Darlinghurst, Sydney 2010, Australia.

³ School of Pathology, University of New South Wales, Sydney 2052, Australia.
^a Author for correspondence. Fax 61-2-8396-5811; e-mail cfuery{at}medau.jnj.com

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 amplification of sequences that lack the RE site.

Methods: Assays 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.

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 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.

Conclusion: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.

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