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

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2010-05, Vol.56 (5), p.814-822
• Main Authors: LUMING ZHOU, PALAIS, Robert A, SMITH, G. Denice, ANDERSON, Daniel, ROWE, Leslie R, WITTWER, Carl T
• Format: Article
• Language: English
• Subjects: Advantages; Alleles; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cancer; Cell Line; Colleges & universities; DNA Primers - genetics; Drug resistance; Efficiency; ErbB Receptors - genetics; Fundamental and applied biological sciences. Psychology; Humans; Hybridization; Investigative techniques, diagnostic techniques (general aspects); Leukemia; Limit of Detection; Lung Neoplasms - genetics; Medical sciences; Melting; Methods; Molecular biophysics; Mutation; Polymerase Chain Reaction - economics; Polymerase Chain Reaction - methods; Proto-Oncogene Proteins B-raf - genetics; Sarcoma; Thyroid; Thyroid Neoplasms - genetics
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2009.142034

Selective amplification of minority alleles is often necessary to detect cancer mutations in clinical samples. 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. 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 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 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. 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