This application note describes how the Agilent 2100 bioanalyzer can be used for the detection of a point mutation in the human prothrombin 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.
The document details a method for detecting the G20210A mutation in the human prothrombin gene, which is associated with an increased risk of venous thrombosis. The study compares traditional agarose gel electrophoresis with microfluidic analysis using the Agilent 2100 bioanalyzer.
| PCR reaction volume | 15 pL |
|---|---|
| DNA concentration per reaction | about 80 ng |
| Primer concentration | 0.33 pmol/pL |
| PCR cycle temperature (denaturation) | 94 °C for 30 s |
| PCR annealing temperature | 52 °C for 30s |
| Restriction digest volume | 20 pL |
| Hind III incubation time | 4 hours |
| Agilent 2100 run time (microfluidic) | within 30 minutes |
| Wild type fragment size | 345 bp |
| Mutant fragment sizes | 822 bp and 23 bp |
The G20210A mutation in the untranslated part of the prothrombin gene.
It offers superior sizing accuracy, quantitation capability, reproducibility, and resolution for closely located DNA bands.