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The Factor II (Prothrombin) G 20210 A Detection and Genotyping August 2 nd, 2010 By Christi Le
Factor II (Prothrombin) G 20210 A 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) G 20210 A Assay A. Extraction- Mag. NA Pure LC DNA Isolation Kit I and instrument (Roche Diagnostics) B. Amplification/ Detection/ Genotyping= factor II (Prothrombin G 20210 A Kit and Light. Cycler 2. 0 instrument. (Roche Diagnostics)
Extraction: Specimen- EDTA whole blood (50 m. L whole blood= 100 m. L purification product) Mag. NA Pure LC Total Nucleic Acid Isolation Kit – large volume 192 Isolation; sample cartridge, elution cartridge, disposable pipette tip tray assembly, and processing cartridge. Mag. NA Pure LC Instrument- automated method DNA I Blood Cell Fast.
Real time PCR (or Amplification or detection) Target- 165 bp fragment of Factor II gene Factor II (Prothrombin) G 20210 A kit- master mix reagents primers and probes) Light. Cycler 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 m. L 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