Search

Objectives: Familial hypercholesterolemia is a monogenic disorder caused by mutations in the LDL receptor ( LDLR ) gene. We observed allelic drop-out during LDLR genotyping and aimed at redesigning mutation detection. Design and methods: The NanoChip microelectronic array technology and PCR restriction fragment length polymorphism analysis were used. Results: Allele drop-out caused false homozygous diagnoses and was overcome using PCR primers without polymorphisms in the primer binding site. Conclusions: This report presents the importance of allele drop-out in LDLR genotyping.

Objectives: To develop and study novel microtiter well based DNA hybridization assays in which the DNA serves as a reporter molecule. Methods: Two hybridization assay configurations are proposed. In configuration A the target DNA is end-labeled with biotin and captured to streptavidin-coated wells. The one strand is removed by NaOH treatment and the other is hybridized with a dATP-tailed oligonucleotide probe. Configuration B involves simultaneous hybridization of heat-denatured target DNA with a biotinylated capture probe (immobilized on streptavidin-coated wells) and a dATP-tailed detection probe. In both configurations the hybrids are reacted with dTTP-tailed luciferase-coding DNA fragment followed by in vitro expression of the DNA on the solid phase. This is accomplished either by a coupled transcription/translation or by sequential transcription and translation reactions optimized separately. Results: The signal-to-background ratios for configuration A at 0.93 fmoles target DNA were 2.6 and 16.7 with the coupled and the separated transcription/translation protocols, respectively. As low as 0.1 fmoles target DNA can be detected with the separate transcription/translation protocol with a signal-to-background ratio of 2.7. The signal-to-background ratios obtained for 0.1 fmoles target DNA with configuration B using the coupled and the separate expression protocols were 2.2 and 4.6, respectively. The average CV was 10%. Conclusion: The expression yield is significantly improved with the separate transcription/translation protocol. Both assay configurations offer high sensitivity and are easily automatable.

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.