TRIP

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

Clinical Chemistry, 2010

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 one 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 Mg(2) concentration and a higher hairpin Tm 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.
Snapback 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 enrichment.