optimization of experimental conditions for rnabased sequencing of mlh1 and msh2 genes the most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic dna as a template for analysis this approach however has cost and sensitivity limitations that can at least in part be overcome by rnabased analysis germline mutations of mlh1 and msh2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary nonpolyposis colorectal cancer hnpcc we compared the analysis of the mlh1 and msh2 genes using mrna and genomic dna as starting material from 21 hnpcc patients all samples were investigated by rtpcr sequencing of cdna and simultaneous sequencing of genomic dna the cdna was generated using specific primers complementary to the ends of mlh1 and msh2 genes respectively mutations in mlh1 and msh2 were detected in 11 out of 21 unrelated patients in 10 out of 11 cases mutations were detected independently of the type of primers used for reverse transcription rt one novel missense mutation k751r in mlh1 was detected using this method one nonsense mutation e205x in msh2 was only detectable when rt was performed using msh2 genespecific primers shorter pcr products indicative of alternatively spliced transcripts were not observed when mlh1 or msh2 specific cdna rt primers were employed to generate template except in one case where exon skipping was observed for exons 9 and 10 in this report we demonstrate that primers specific for rt of mlh1 and msh2 are crucial for increasing the sensitivity of cdna analysis dna sequencing using rna as a basis for template construction may be a valuable and economical alternative to genomic dna sequencing