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The Factor II ( Prothrombin ) G20210A Detection and Genotyping

The Factor II ( Prothrombin ) G20210A Detection and Genotyping. August 2 nd , 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.

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The Factor II ( Prothrombin ) G20210A Detection and Genotyping

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

  2. 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.

  3. 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)

  4. 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.

  5. 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.

  6. 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

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