Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment

E. Castellanos-Rizaldos, Pingfang Liu, Coren A. Milbury, Minakshi Guha, Angela Brisci, Laura Cremonesi, Maurizio Ferrari, Harvey Mamon, G. Mike Makrigiorgos

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 (co-amplification 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 (Tm) 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 Tms. 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 Tms can be mutation enriched via TT-COLD-PCR provided that their Tms 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.

Original languageEnglish
Pages (from-to)1130-1138
Number of pages9
JournalClinical Chemistry
Volume58
Issue number7
DOIs
Publication statusPublished - Jul 2012

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Denaturation
Amplification
Polymerase Chain Reaction
Mutation
Temperature
DNA
Thermal cycling
Tumors
Neoplasms

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Castellanos-Rizaldos, E., Liu, P., Milbury, C. A., Guha, M., Brisci, A., Cremonesi, L., ... Makrigiorgos, G. M. (2012). Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment. Clinical Chemistry, 58(7), 1130-1138. https://doi.org/10.1373/clinchem.2012.183095

Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment. / Castellanos-Rizaldos, E.; Liu, Pingfang; Milbury, Coren A.; Guha, Minakshi; Brisci, Angela; Cremonesi, Laura; Ferrari, Maurizio; Mamon, Harvey; Makrigiorgos, G. Mike.

In: Clinical Chemistry, Vol. 58, No. 7, 07.2012, p. 1130-1138.

Research output: Contribution to journalArticle

Castellanos-Rizaldos, E, Liu, P, Milbury, CA, Guha, M, Brisci, A, Cremonesi, L, Ferrari, M, Mamon, H & Makrigiorgos, GM 2012, 'Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment', Clinical Chemistry, vol. 58, no. 7, pp. 1130-1138. https://doi.org/10.1373/clinchem.2012.183095
Castellanos-Rizaldos E, Liu P, Milbury CA, Guha M, Brisci A, Cremonesi L et al. Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment. Clinical Chemistry. 2012 Jul;58(7):1130-1138. https://doi.org/10.1373/clinchem.2012.183095
Castellanos-Rizaldos, E. ; Liu, Pingfang ; Milbury, Coren A. ; Guha, Minakshi ; Brisci, Angela ; Cremonesi, Laura ; Ferrari, Maurizio ; Mamon, Harvey ; Makrigiorgos, G. Mike. / Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment. In: Clinical Chemistry. 2012 ; Vol. 58, No. 7. pp. 1130-1138.
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AU - Brisci, Angela

AU - Cremonesi, Laura

AU - Ferrari, Maurizio

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AB - 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 (co-amplification 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 (Tm) 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 Tms. 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 Tms can be mutation enriched via TT-COLD-PCR provided that their Tms 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.

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