Temperature-Tolerant COLD-PCR Reduces Temperature Stringency and Enables Robust Mutation Enrichment

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2012-07, Vol.58 (7), p.1130-1138
• Main Authors: CASTELLANOS-RIZALDOS, E, PINGFANG LIU, MILBURY, Coren A, GUHA, Minakshi, BRISCI, Angela, CREMONESI, Laura, FERRARI, Maurizio, MAMON, Harvey, MAKRIGIORGOS, G. Mike
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Brain Neoplasms - genetics; Cancer; Cell Line, Tumor; Cold; Colorectal Neoplasms - genetics; Deoxyribonucleic acid; Detection limits; DNA; DNA - analysis; DNA - genetics; DNA Mutational Analysis - methods; Fundamental and applied biological sciences. Psychology; Glioblastoma - genetics; Humans; Investigative techniques, diagnostic techniques (general aspects); Lung Neoplasms - genetics; Male; Medical sciences; Methods; Microbiology; Molecular biophysics; Mutation; Nucleic Acid Denaturation; Plasma; Polymerase Chain Reaction - methods; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins p21(ras); ras Proteins - genetics; Sarcoma; Temperature; Tumor Suppressor Protein p53 - genetics
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2012.183095

Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. A single thermocycling program with a denaturation-temperature window of 2.5-3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.