title: Sensitivity and specificity of denaturing high-pressure liquid chromatography for unknown protein C gene mutations.
abstract: Screening methods for unknown DNA sequence variations are laborious, expensive, and relatively insensitive. To evaluate the sensitivity and specificity of denaturing high-pressure liquid chromatography (DHPLC) screening for unknown protein C gene (PROC) mutations, we studied 31 PROC-deficient patients. Eleven amplimers containing 4 kb of the PROC gene and spanning all exons, splice junctions, and the putative promoter and 3'-untranslated regions were amplified by PCR for each patient. Each amplimer (n = 341) was sequenced with a fluorescence-based method, and screened by DHPLC. Sequencing identified 10 unique mutations and three polymorphisms. Combining all mutations and polymorphisms, 227 amplimers were homozygous wildtype, and 63 and 51 were heterozygous and homozygous mutant, respectively. DHPLC screening correctly identified all amplimers (100% sensitivity and specificity). DHPLC is a rapid, automated, sensitive and specific screening method for unknown mutations within the PROC gene, and may be a useful screening method for unknown mutations within other genes.
authors.affiliation: Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Taliani
authors.forename: S C
authors.initials: SC
authors.lastname: Roberts
authors.forename: B A
authors.initials: BA
authors.lastname: Dukek
authors.forename: R K
authors.initials: RK
authors.lastname: Pruthi
authors.forename: W L
authors.initials: WL
authors.lastname: Nichols
authors.forename: J A
authors.initials: JA
authors.lastname: Heit
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons / Q000235:genetics; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics*; D020125:Mutation, Missense / Q000235:genetics; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic / Q000235:genetics; D011486:Protein C / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D011486:Protein C; D022821:RNA Splice Sites