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Clinical Chemistry 56: 814-822, 2010. First published March 18, 2010; 10.1373/clinchem.2009.142034
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(Clinical Chemistry. 2010;56:814-822.)
© 2010 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR

Luming Zhou1,a, Robert A. Palais2, G. Denice Smith3, Daniel Anderson3, Leslie R. Rowe3 and Carl T. Wittwer1,3,a

1 Departments of Pathology and 2 Mathematics, University of Utah Health Sciences Center, Salt Lake City, UT; 3 ARUP Laboratories and Institute for Clinical and Experimental Pathology, Salt Lake City, UT.

aAddress 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{at}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{at}path.utah.edu.

Background: Selective amplification of minority alleles 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 1 PCR primer hybridizes to the variable locus of its extension product to produce a hairpin that selectively enriches mismatched alleles. Genotyping performed after rapid-cycle PCR by melting of the secondary structure identifies different variants by the hairpin melting temperature (Tm). Needle aspirates of thyroid tissue (n = 47) and paraffin-embedded biopsy samples (n = 44) were analyzed for BRAF (v-raf murine 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 [epidermal growth factor receptor (erythroblastic leukemia viral (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 selective amplification of mismatched alleles. A low Mg2+ concentration and a higher hairpin Tm relative to the extension temperature also improved the detection limit of mismatched alleles. The detection limit was 0.1% for BRAF p.V600E and 0.02% for EGFR exon 19 in-frame deletions. Snapback and dual hybridization probe methods for allele quantification of the thyroid samples correlated well (R2 = 0.93) with 2 more BRAF mutations (45 and 43, respectively, of 91 samples) detected after snapback enrichment. Different EGFR in-frame deletions in the lung samples produced different hairpin Tms.

Conclusions: Use of snapback primers for enrichment and detection of minority alleles is simple, is inexpensive to perform, and can be completed in a closed tube in <25 min.