Abstract 2118: Rare allele enrichment by ARMS combined with probe base genotyping techniques

Abstract

Abstract Mutation detection plays a key role in several medical areas for diagnosis, treatment and prognosis. Additionally, the ability to detect low level mutations among mostly wild-type DNA is critical to areas such as cancer detection, prenatal testing and infectious diseases. For example, low-level (<10%) cancer mutations can not be sequenced by standard PCR or genotyping techniques. Many moderate- to high-selectivity PCR methods have been developed over the past two decades to enrich minority alleles for known mutations. One of the most widely used approaches is the amplification refractory mutation system (ARMS) which relies on the use of 3’ end terminal nucleotide mismatch to enhance the minor allele. The mutation detection sensitivity ranges from 1:10 to 1:1000. Probe based genotyping techniques such as fluorescent labeled probes, unlabeled probes and Snapback primers depend on short oligonucleotides that hybridize to the template for genotyping. We have combine allele enrichment by ARMS and probe based genotyping techniques to enhance allele discrimination. We designed a 3’ end blocked oligonucleotides to form a perfect match with the wild- type allele (mismatched to other alleles). Therefore, the probe blocks the primer from annealing to the wild-type template, while simultaneously permitting the primer to anneal to, and amplify, the mutant template. Wild-type amplification is completely blocked, and the mutation is enriched. The sensitivity of unlabeled probe-ARMS mutation enrichment detection is 1:30,000. Combining ARMS enrichment of minority alleles and snapback primers has the additional benefit that only two PCR primers are required, with the addition of a short tail of nucleotides on one primer that results in an intra-molecular hybridization probe. The sensitivity of snapback-ARMS mutation enrichment detection is 1:60,000. Fourty-four patients with paired thyroid biopsies and needle samples and 3 additional patients with only needle samples were tested for the BRAF mutation p.V600E (c.1799 T>A) by dual hybridization probes. A blinded test using unlabeled probe-ARMS and Snapback-ARMS enrichment techniques were compared to the dual hybridization probe method. All samples were concordant (47 positive and 42 negative) except 4 samples were positive using unlabeled probe-ARMS and Snapback-ARMS but were negative using hybridization probes. Minority allele enrichment using ARMS combined with probe base genotyping methods increases the mutation detection sensitivity 100 times or more. ARMS combined with an unlabeled probe or a snapback primer is an inexpensive and rapid enrichment technique. No separation, purification or addition steps that increase risk of contamination are necessary. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2118.