title: Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: E
authors.initials: E
authors.lastname: Castellanos-Rizaldos
authors.forename: Pingfang
authors.initials: P
authors.lastname: Liu
authors.forename: Coren A
authors.initials: CA
authors.lastname: Milbury
authors.forename: Minakshi
authors.initials: M
authors.lastname: Guha
authors.forename: Angela
authors.initials: A
authors.lastname: Brisci
authors.forename: Laura
authors.initials: L
authors.lastname: Cremonesi
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.forename: Harvey
authors.initials: H
authors.lastname: Mamon
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D001932:Brain Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005909:Glioblastoma / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D008297:Male; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins