Agilent Detection of a point mutation in the prothrombin gene with the Agilent 2100 bioanalyzer Application Note说明书用户手册.PDF

Detection of a point mutation in the prothrombin gene with the Agilent 2100 bioanalyzer Application Note Marton Andrasfalvy Ph.D. Enikõ Kámory Bela Csokay Ph.D. Abstract This Application Note describes how the Agilent 2100 bioanalyzer can be used for the detection of a point mutation in the human prothrom- bin gene. The prothrombin protein plays a key role in blood clotting. A single nucleotide polymorphism (SNP), i.e. a point mutation in the prothrombin gene results in a common hereditary predisposition to venous thrombosis. We present a convenient and reliable mutation detection method by a PCR-based assay including a specific restriction digest (PCR-RFLP). Due to its high sensitivity and resolution, the applied microfluidic assay proves to be extremely suitable for detec- tion of the obtained PCR products and restriction fragments. The developed method is suitable for the workflow at Laborigo molecular diagnostic laboratory. Introduction fied. The two fragments potential- 30 s, 52 ºC for 30 s, 72 ºC for 30 s ly generated in this PCR-RFLP and a final extension step of 72 ºC The G20210A mutation in the protocol are close in size. There- for 5. The reverse primer’s last untranslated part of the prothrom- fore, we did a performance com- nucleotides hybridize just adjacent bin gene results in an elevated parison between traditional with the potential point mutation serum prothrombin level and an agarose slab gel method and in prothrombin DNA-strand. The increased risk for venous throm- Lab-on-a-Chip technology using a nucleotide marked with an aster- bosis. Individuals heterozygous for microcapillary electrophoresis isk in the reverse pri