Τίτλος:
Development of a universal chemiluminometric genotyping method for high-throughput detection of 7 LDLR gene mutations in greek population
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
Objectives: Familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor (LDLR) gene. We report the application of a universal method with high allele discrimination properties to the simultaneous genotyping of 7 LDLR mutations in Greeks, in dry-reagent format. Design and methods: We genotyped mutations C858A, C939A, G1285A, T1352C, G1646A, G1775A, C/T81G. Unpurified amplicons from a multiplex PCR that produced fragments encompassing all 7 mutations were subjected to probe extension reactions in the presence of fluorescein-modified dCTP, and a microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents and assigned genotypes by the signal ratio of normal-to-mutant-specific probe. Results: We standardized the method and optimised all steps for specificity. The method was validated by genotyping blindly 119 (833 genotypings). Results were fully concordant with other methods used as standards. Conclusions: This method is accurate, simple, rapid and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations. © 2007 The Canadian Society of Clinical Chemists.
Συγγραφείς:
Glynou, K.
Laios, E.
Drogari, E.
Tsaoussis, V.
Περιοδικό:
Clinical Biochemistry
Λέξεις-κλειδιά:
adenine; cytosine; deoxycytidine triphosphate; DNA polymerase; fluorescein; guanine; horseradish peroxidase; low density lipoprotein receptor; reagent; thymine, accuracy; allele; amplicon; article; chemoluminescence; conjugation; familial hypercholesterolemia; freeze drying; gene; gene mutation; gene probe; genotype; Greece; high throughput screening; human; ldlr gene; major clinical study; microtiter plate assay; multiplex polymerase chain reaction; mutational analysis; priority journal; probe extension reaction; sensitivity and specificity; technique; validity, Chemiluminescent Measurements; DNA Mutational Analysis; European Continental Ancestry Group; Genotype; Greece; Humans; Mutation; Polymerase Chain Reaction; Receptors, LDL; Reproducibility of Results
DOI:
10.1016/j.clinbiochem.2007.12.016
