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COLD-PCR Enriches Low-Level Variant DNA Sequences and Increases the Sensitivity of Genetic Testing

  • Elena Castellanos-Rizaldos
  • Coren A. Milbury
  • Minakshi Guha
  • G. Mike Makrigiorgos
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1102)

Abstract

Detection of low-level mutations is important for cancer biomarker and therapy targets discovery, but reliable detection remains a technical challenge. The newly developed method of CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) helps to circumvent this issue. This PCR-based technology preferentially enriches minor known or unknown variants present in samples with a high background of wild type DNA which often hampers the accurate identification of these minority alleles. This is a simple process that consists of lowering the temperature at the denaturation step during the PCR-cycling protocol (critical denaturation temperature, T c) and inducing DNA heteroduplexing during an intermediate step. COLD-PCR in its simplest forms does not need additional reagents or specific instrumentation and thus, can easily replace conventional PCR and at the same time improve the mutation detection sensitivity limit of downstream technologies. COLD-PCR can be applied in two basic formats: fast-COLD-PCR that can enrich T m-reducing mutations and full-COLD-PCR that can enrich all mutations, though it requires an intermediate cross-hybridization step that lengthens the thermocycling program. An improved version of full-COLD-PCR (improved and complete enrichment, ice-COLD-PCR) has also been described. Finally, most recently, we developed yet another form of COLD-PCR, temperature-tolerant-COLD-PCR, which gradually increases the denaturation temperature during the COLD-PCR reaction, enriching diverse targets using a single cycling program. This report describes practical considerations for application of fast-, full-, ice-, and temperature-tolerant-COLD-PCR for enrichment of mutations prior to downstream screening.

Key words

Coamplification at lower denaturation temperature (COLD-PCR) Mutation detection Low-level mutations Cancer Diagnosis Prognosis Therapy targets 

Notes

Acknowledgment

This work was supported by the Innovative Molecular Analysis Technologies Program of the NCI, grants CA-111994 and CA-151164 (G.M.M.). The contents of this manuscript do not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Elena Castellanos-Rizaldos
    • 1
  • Coren A. Milbury
    • 2
  • Minakshi Guha
    • 3
  • G. Mike Makrigiorgos
    • 2
  1. 1.Division of DNA Repair and Genome Stability, Department of Radiation OncologyDana-Farber Cancer Institute, Harvard Medical SchoolBostonUSA
  2. 2.Department of Radiation OncologyDana-Farber Cancer Institute, Harvard Medical SchoolBostonUSA
  3. 3.Divisions of DNA Repair and Genome Stability and Medical Physics and Biophysics, Department of Radiation OncologyDana-Farber Cancer Institute, Harvard Medical SchoolBostonUSA

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