title: Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis.
abstract: We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.
authors.affiliation: Klinisch-Chemisches Zentrallabor, Universitätskliniken des Saarlandes, Homburg, Germany. kchjgei@med-rz.uni-sb.de
authors.forename: J
authors.initials: J
authors.lastname: Geisel
authors.forename: T
authors.initials: T
authors.lastname: Walz
authors.forename: M
authors.initials: M
authors.lastname: Bodis
authors.forename: M
authors.initials: M
authors.lastname: Nauck
authors.forename: K
authors.initials: K
authors.lastname: Oette
authors.forename: W
authors.initials: W
authors.lastname: Herrmann
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D018807:Polymorphism, Single-Stranded Conformational; D011973:Receptors, LDL / Q000235:genetics*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D011973:Receptors, LDL