Digital PCR goes COLD: Application of COLD-PCR in digital format enables quantitative and multiplexed mutation scanning

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SELECTBIO

As currently applied, digital PCR can only be used to detect known mutations at single sequence positions, for 'hotspot' mutations. Here we adapt digital droplet PCR to operate under COLD-PCR conditions, thereby preferentially amplifying mutation-containing sequences in droplets. COLD-PCR enables amplicons with mutations at any position to be amplified preferentially, while wild-type samples are preferentially suppressed. The preferentially amplified sequences can subsequently be sequenced. We validate the merging of technologies in serially diluted TP53 and KRAS mutated sequences. By merging COLD-PCR and digital -PCR technologies we enable digital mutation scanning, a new frontier for digital-PCR. A single reaction can now interrogate numerous mutations, and replaces multiple individual digital-PCR reactions.