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.

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

“…However, the sensitivity of HRM analysis of standard PCR products has generally been found to be limited to mutant alleles present at 5%-10% in a background of wild type DNA (Krypuy, et al, 2007;Krypuy, et al, 2006;Polakova, et al, 2008;Takano, et al, 2007). Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that significantly increases the sensitivity of mutation detecting assays without adding to the costs and complexity of the experiment (Li, et al, 2008).…”

“…In general, mutation enrichment can be expected to be 5-8 fold for melting temperature retaining mutations and 3-5 fold for melting temperature increasing mutations when using full COLD-PCR as opposed to standard PCR. A thorough description of the differences between fast and full COLD-PCR can be found in (Li, et al, 2008). In the human genome approximately 70% of all encountered mutations, and 84% of the somatic mutations found in colon cancer, are melting temperature decreasing (Li, et al, 2008).…”

Mutant Enrichment with 3′-Modified Oligonucleotides

“…The co-amplification at lower denaturation temperature PCR technique utilizes differences in the T m s of PCR amplicons between wildtype and mutant (or mismatched) DNA sequences. 10 This technique simply adjusts the denaturation temperatures during the PCR cycle and requires no additional steps or costs. An advantage of this technique is that multiple targets within the amplified region can be enriched.…”

“…These recent enrichment PCR techniques include thermostable restriction endonuclease-mediated selective PCR, 6 PCR clamping mediated by peptide nucleic acid (PNA) 7,8 or locked nucleic acid (LNA), 9 and co-amplification at lower denaturation temperature PCR. 10 Each technique has its own strengths and limitations in regard to the cost, availability, or enrichment efficiency.…”

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

“…In the current study, co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) was employed as a novel modification of the conventional PCR method so as to go beyond the limitations currently encountered in detecting mutations (Li et al, 2008). COLD-PCR, combined with unlabeled-probe HRM, an integration of our previous approaches, was thus developed for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of PA, thereby expanding the scope of the modular diagnosis application in the clinic (Wei et al, 2012).…”