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Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

Abstract

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

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Figure 1: Description of COLD-PCR for an example 167-bp TP53 sequence.
Figure 2: COLD-PCR improves the sensitivity of Sanger dideoxy-terminator sequencing.
Figure 3: Examples of low-level mutations in solid tumor clinical samples, previously 'invisible' in Sanger dideoxy sequencing, that now become detectable via COLD-PCR.
Figure 4: COLD-PCR improves the sensitivity of pyrosequencing.

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Acknowledgements

We gratefully acknowledge the assistance of A. Brown at the Harvard Partners Center for Genetics and Genomics High Throughput Genotyping Facility and of M. Miri, F. Haluska and P. Janne in obtaining specimens from the Massachusetts General Hospital Tumor Bank and Dana Farber Cancer Institute. We also acknowledge B. Price and A. D'Andrea for valuable comments on the manuscript. This work was supported by training grant 5 T32 CA09078 (J.L.) and US National Institutes of Health grants CA111994-01 and CA115439-01.

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J.L. and L.W., experimental design; H.M. and M.H.K., clinical considerations and rationale; R.B., modeling; G.M.M., project setup, experimental design and manuscript preparation.

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Correspondence to G Mike Makrigiorgos.

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Supplementary Figs. 1–8 and Supplementary Table 1 (PDF 883 kb)

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Li, J., Wang, L., Mamon, H. et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14, 579–584 (2008). https://doi.org/10.1038/nm1708

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