Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR Luming Zhou, 1* Robert A. Palais, 2 G. Denice Smith, 3 Daniel Anderson, 3 Leslie R. Rowe, 3 and Carl T. Wittwer 1,3* BACKGROUND: Selective amplification of minority al- leles is often necessary to detect cancer mutations in clinical samples. METHODS: Minor-allele enrichment and detection were performed with snapback primers in the presence of a saturating DNA dye within a closed tube. A 5' tail of nucleotides on one PCR primer hybridizes to the vari- able locus of its extension product to produce a hairpin that selectively enriches mismatched alleles. Genotyp- ing performed after rapid-cycle PCR by melting of the secondary structure identifies different variants by the hairpin melting temperature (T m ). Needle aspirates of thyroid tissue (n = 47) and paraffin-embedded biopsy samples (n = 44) were analyzed for BRAF (v-raf mu- rine sarcoma viral oncogene homolog B1) variant p.V600E, and the results were compared with those for dual hybridization probe analysis. Needle aspirates of lung tumors (n = 8) were analyzed for EGFR [epider- mal growth factor receptor (erythroblastic leukemia vi- ral (v-erb-b) oncogene homolog, avian)] exon 19 in- frame deletions. RESULTS: Use of 18-s cycles and momentary extension times of “0 s” with rapid-cycle PCR increased the selec- tive amplification of mismatched alleles. A low Mg 2+ concentration and a higher hairpin T m relative to the extension temperature also improved the detection limit of mismatched alleles. The detection limit was mutant–wild type ratio of 1:1000 for BRAF p.V600E and 1:5000 for EGFR exon 19 in-frame deletions. Snap- back and dual hybridization probe methods for allele quantification of the thyroid samples correlated well (r = 0.93) with 2 more BRAF mutations (45 and 43, respectively, of 91 samples) detected after snapback en- richment. Different EGFR in-frame deletions in the lung samples produced different hairpin T m s. CONCLUSIONS: Use of snapback primers for enrichment and detection of minority alleles is simple, is inexpen- sive to perform, and can be completed in a closed tube in 25 min. © 2010 American Association for Clinical Chemistry Since the sequencing of the human genome and the development of high-throughput genotyping tech- niques, many genes that are associated with disease through single-base and other mutations have been identified. For example, both prenatal and cancer diag- nostics previously performed by chromosomal or phe- notype analysis are now done at the nucleotide level (1–3 ). Mutations in DNA circulating in the plasma can serve as biomarkers of early tumor development and the potential response to treatment (4, 5 ). A somatic mutation of the BRAF 4 (v-raf murine sarcoma viral oncogene homolog B1) gene, c.1799 TA, which en- codes the substitution p.V600E, is the most common change in papillary thyroid carcinoma (6), having occurred in 80% of the cases studied (7). In lung cancer, in-frame deletions of exon 19 in the EGFR gene [epidermal growth factor receptor (erythroblastic leu- kemia viral (v-erb-b) oncogene homolog, avian)], which encodes the epidermal growth factor reporter, can confer either a positive therapeutic response to ty- rosine kinase inhibitors or drug resistance (8, 9 ). In the early and posttreatment stages of cancer, the proportions of mutant alleles in clinical samples are typically low compared with the wild-type allele. Even after amplification by standard PCR, concentrations of the minority allele may not be sufficient to permit genotyping or sequencing (10 –13 ). There is a wide range of PCR-based techniques for enriching the pro- portion of minority alleles and mutations in a sample. When the genotype of the mutation is unknown, 1 Departments of Pathology and 2 Mathematics, University of Utah Health Sci- ences Center, Salt Lake City, UT; 3 ARUP Laboratories and Institute for Clinical and Experimental Pathology, Salt Lake City, UT. * Address correspondence to: L.Z. at Department of Pathology, University of Utah Medical School, 50 N. Medical Dr., Salt Lake City, Utah 84132. Fax +801-581- 6001; e-mail luming.zhou@path.utah.edu. C.T.W. at Department of Pathology, University of Utah Medical School, 50 N. Medical Dr., Salt Lake City, Utah 84132. Fax +801-581-6001; e-mail carl.wittwer@path.utah.edu. Received December 14, 2009; accepted February 24, 2010. Previously published online at DOI: 10.1373/clinchem.2009.142034 4 Human genes: BRAF, v-raf murine sarcoma viral oncogene homolog B1; EGFR, epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) onco- gene homolog, avian). Clinical Chemistry 56:5 000 – 000 (2010) Molecular Diagnostics and Genetics 1 http://www.clinchem.org/cgi/doi/10.1373/clinchem.2009.142034 The latest version is at Papers in Press. Published March 18, 2010 as doi:10.1373/clinchem.2009.142034 Copyright (C) 2010 by The American Association for Clinical Chemistry