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

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 (T-m). 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 T-m 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 (R-2 = 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 T(m)s. 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. (C) 2010 American Association for Clinical Chemistry