COLD-HRM: a combination of methods to infer the nature of somatic mutations

High Resolution Melting HRM measures DNA melting patterns to detect mutations Hitherto HRM has not been used to report the nature of the mutation We reasoned that in conditions with a minor allele e g allelic loss and somatic mutation CO amplification at Lower Denaturation temperature COLD PCR could change HRM patterns allowing i detection of Loss of Heterozygosity LOH and ii discrimination of somatic mutations from germline variants For LOH we tested paired tumour normal samples from cases of colorectal cancer CRC which were heterozygous for the SMAD SNP rs and case which was homozygous wild type To discriminate somatic from germline mutation we tested tumour from CRCs with somatic mutation in codon POLE paired tumour normal tissue from CRCs heterozygous for POLESNP rs and matched normal samples wild type All samples underwent both the standard and COLD PCR followed by HRM analysis In tumour samples which were heterozygous for the SMAD SNP HRM patterns changed following COLD PCR suggesting LOH Changes in HRM patterns were also seen in tumours with POLE codon mutation suggesting somatic rather than germline mutation No change was seen in the tumour and matched normal samples heterozygous for POLESNP rs We conclude that combining COLD PCR with HRM COLD HRM allows inference of LOH and somatic sequence mutation without the requirement for matched normal DNA