The Factor II (Prothrombin) G20210A Detection and Genotyping August 2nd, 2010 By Christi Le

Factor II (Prothrombin) G20210A Most common inherited coagulation disorder in U.S. Autosomal dominant inheritance. Inherited condition Third most common cardiovascular disease. Single point mutation (G to A at position 20210) 2-5% in Caucasians 0.3% in African Americans Heterozygous= 2-4 fold increased risk of thrombosis. Homozygous= 80 fold increased risk of thrombosis. Treatment= clinical circumstances and may include anticoagulation therapy.

Factor II (Prothrombin) G20210A Assay Extraction- MagNA Pure LC DNA Isolation Kit I and instrument (Roche Diagnostics) Amplification/ Detection/ Genotyping= factor II (Prothrombin G20210A Kit and LightCycler 2.0 instrument. (Roche Diagnostics)

Extraction: Specimen- EDTA whole blood (50 mL whole blood= 100 mL purification product) MagNA Pure LC Total Nucleic Acid Isolation Kit – large volume 192 Isolation; sample cartridge, elution cartridge, disposable pipette tip tray assembly, and processing cartridge. MagNA Pure LC Instrument- automated method DNA I Blood Cell Fast.

Real time PCR (or Amplification or detection) Target- 165bp fragment of Factor II gene Factor II (Prothrombin) G20210A kit- master mix reagents primers and probes) LightCycler 2.0 instrument- 32 cycles in 30 minutes. There are 3 cycle temperature- denaturation 95 degrees Celsius, annealing 60 degrees Celsius, and elongation 72 degrees Celsius. Thermal cycler: heat/ambient air cycle. Reaction tube: 20 mL glass capillary tube.

Summary: Erroneous results: -patient sample with elevated WBC -false positive (3 rare mutations piece same mutation probe) Limitations: high conc. of heparin might interfere with the PCR/instruments and technical process. Overall, it is a very clever system and also an important role in molecular diagnostic clinical lab