Sensitivity and specificity of denaturing high-pressure liquid chromatography for unknown protein C gene mutations

Screening methods for unknown DNA sequence variations are laborious, expens ive, and relatively insensitive. To evaluate the sensitivity and specificit y of denaturing high-pressure liquid chromatography (DHPLC) screening for u nknown protein C gene (PROC) mutations, we studied 31 PROC-deficient patien ts. Eleven amplimers containing 4 kb of the PROC gene and spanning all exon s, splice junctions, and the putative promoter and 3'-untranslated regions were amplified by PCR for each patient. Each amplimer (a = 341) was sequenc ed with a fluorescence-based method, and screened by DHPLC, Sequencing iden tified 10 unique mutations and three polymorphisms. Combining all mutations and polymorphisms, 227 amplimers were homozygous wild-type, and 63 and 51 were heterozygous and homozygous mutant, respectively, DHPLC screening corr ectly identified all amplimers (100% sensitivity and specificity). DHPLC is a rapid, automated, sensitive and specific screening method for unknown mu tations within the PROC gene, and may be a useful screening method for unkn own mutations within other genes.