Light cycler real time PCR teknolojisi ile faktör V geninde yeni mutasyon taraması
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Date
2009
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Biyoteknoloji Enstitüsü
Abstract
The risk of venous thrombosis is increased when the hemostatic balance between pro- and anticoagulant forces is shifted in favor of coagulation. If this is caused by an inherited defect, the resulting hypercoagulablestate conveys a lifelong increased risk of thrombosis. Mutations in the genes coding for proteins in the coagulation cascade were found to be associated with increased risk for venous thrombosis. Dahlbäck et al. described a predisposition to thrombosis caused by a poor anticoagulant response to activated protein C (APC). A year later Bertina et al., reported that a point mutation in exon 10 of the gene encoding blood coagulation factor V.Real Time PCR is a system, which analyses a mutation Factor V Leiden. In this technique hybridization probe can be used to detect and monitor single nucleotide mutation. The probe has 2 part and its specific to mutation. With the melting curve analysis the hybridization probes melt off, two fluorescent dyes are no longer in close proximity and the fluorescence will degrees. The resulting melting peaks allow discrimination between the homozygous as well as the heterozygous genotype. The anchor probe has a higher Tm than the mutation probe. During the experiment tm degree of the unknown samples may vary ±2ºC from the tm degree of positive control. Differences in base composition, nucleotide position, and nearest-neighbor environments all affect Tm.There were 4100 diagnostic samples for thrombophilia screening starting 2002 in our department. We found 13 patients with a variation of Tm degree more than ±2ºC. Results of DNA sequencing showed only FVL.In order to delineate the reason for the artifact of the Tm degree, DNA of the patients were isolated again and the study was repeated by LC and sequencing. The shift of the LC experiment was within normal range indicating that they are FVL.Our study showed that DNA quality and quantity is important and needs to be analyzed prior to laboratory procedures and archival and not well-kept DNA samples may cause the same variation, which causes an artifact
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Keywords
Light cycler, PCR teknolojisi, V geni, mutasyon