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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 …