DHPLC analysis of HNPCC: a rapid sensitive exon screen of hMLH1 and hMSH2. J.F. Harvey¹, S.P. Haynes¹, D.M. Eccles². 1) Wessex Regional Genetics Lab, Salisbury District Hospital, Salisbury, Wiltshire, United Kingdom; 2) Wessex Clinical Genetics Service, Level G, Princess Anne Hospital, Coxford Road, Southampton, Hants, SO16 5YA, United Kingdom.
   hMLH1 and hMSH2 are two of the four mismatch repair genes mutated in hereditary non-polyposis colorectal cancer (HNPCC) and account for two-thirds of affected families. They have complex genomic structures (19 and 16 exons respectively) with diverse mutational spectra and few common mutations. We have developed a rapid highly sensitive screen of coding exons and splice junctions using a 96 well microtitre plate format with multichannel pipette transfer and intron derived primers. Mutations are detected using denaturing high performance liquid chromatography at two melt temperatures (derived using Transgenomic Wavemaker software) for PCR fragments amplified with AmpliTaq Gold. We identified all 29 unique, mutations/ polymorphisms (14 amino acid changes, 2 splice site mutations, 2 insertions and 11 deletions) previously found in our family samples by combined SSCP/heteroduplex analysis on silver stained polyacrylamide gels. The resolution of DHPLC heteroduplex analysis suggests that sensitivity of this approach is well in excess of the original gel based method.