Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

Blasczyk, Rainer; Ritter, Markus; Thiede, Christian; Wehling, J.; Hintz, G.; Neubauer, Andreas; Riess, H.

doi:10.1055/s-0038-1650362

Cited by 19 publications

(8 citation statements)

References 3 publications

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“…Genomic DNA was prepared by the DNA Zol method. 18 Samples were investigated with 2 polymerase chain reaction (PCR) amplifications, one amplifying the wild-type and the other amplifying the factor V point mutation. Each PCR mix included a primer pair that amplified a fragment of the human growth hormone gene (internal positive amplification control 3,18 ).…”

Section: Laboratory Parametersmentioning

confidence: 99%

Poor Response to Activated Protein C as a Prominent Risk Predictor of Advanced Atherosclerosis and Arterial Disease

Kiechl¹,

Muigg²,

Santer³

et al. 1999

Circulation

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Section: Laboratory Parametersmentioning

confidence: 99%

Poor Response to Activated Protein C as a Prominent Risk Predictor of Advanced Atherosclerosis and Arterial Disease

Kiechl¹,

Muigg²,

Santer³

et al. 1999

Circulation

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“…Additional mismatches were included towards the 3'-ends of the reverse primers in order to increase the specificity of the reaction (Table 1). Primers directed at the factor V gene were taken from previously published work (25), and those for the prothrombin gene were specifically designed for this study. Exon 10 in the factor V gene and the 3'-UTR region of the prothrombin gene were amplified from normal individuals together with both homozygotes and heterozygotes for the R506Q and the G20210A mutations.…”

Section: Resultsmentioning

confidence: 99%

“…Several novel PCR strategies have been described to detect the latter mutations individually (7,11,(18)(19)(20)(21)(22)(23)(24)(25). The rate-limiting step in many of these protocols is DNA isolation.…”

Section: Discussionmentioning

confidence: 99%

Double Fluorescent-amplification Refractory Mutation Detection (dF-ARMS) of the Factor V Leiden and Prothrombin Mutations

Maher¹,

Crowley²,

Cullen³

et al. 1999

Thromb Haemost

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SummarySimultaneous fluorescent [F] detection of the factor V Leiden (G1691A) and the prothrombin 3’-untranslated region (G20210A) mutations were performed in a single tube polymerase chain reaction (PCR). Amplification refractory mutation detection system (ARMS) formed the basis of this assay design. Fluorescent-labelled primers incorporated into amplicons during the reaction facilitated detection directly by GeneScan analysis without further manipulation. To test the efficacy of this double [F]-ARMS (dF-ARMS) method, 48 patients with unexplained thrombotic tendencies were investigated for their factor V Leiden and prothrombin genotypes. These results corresponded exactly with data achieved using the more conventional methods of restriction fragment length polymorphism (RFLP)-PCR and direct DNA sequencing. Three out of the 48 patients in this group were found to be compound heterozygotes.

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“…In the past, many different PCR-based assays have been used to identify FV Leiden and other SNPs (10)(11)(12). The majority of these assays involve extensive and time-consuming post-PCR steps which are not easily adaptable for either multiple gene analysis or high throughput.…”

Section: Introductionmentioning

confidence: 99%

Factor V Leiden and Factor V R2 Allele: High-throughput Analysis and Association with Venous Thromboembolism

Benson¹,

Ellingsen²,

El-Jamil³

et al. 2001

Thromb Haemost

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SummaryThrombophilia is a multigenic disease in which the combination of genetic polymorphisms increases the risk of deep vein thrombosis (DVT). The rapid identification of these genetic combinations requires high-throughput analysis of single nucleotide polymorphisms (SNPs). The TaqMan® fluorogenic 5’→3’ nuclease assay (PE/Applied Bio-systems, Foster City, CA) with custom-designed primers, probes and controls has provided a highly efficient platform for high throughput. This assay was used to rapidly detect two SNPs, FV Leiden (G1691A) and FV A4070G (R2 allele), in a study of 6295 subjects. With one thermal cycler, we completed sample set-up, PCR and analysis on 84 samples in 3 h with an additional 12 wells containing 4 “no template controls” (NTC), 4 “allele-1 controls”, and 4 “allele-2 controls” in a 96-well plate. When additional thermal cyclers were used and more assays were set up while the initial sets of reactions were in the PCR machines, the output could correspondingly be increased. The TaqMan® assay was extremely accurate, avoided contamination by using uracil-N-glycolase (UNG) in a single, closed tube, and offered the possibility for additional automation with robotic equipment to implement the PCR. This TaqMan® assay facilitates high throughput to screen large populations quickly and economically while utilizing a simple protocol that requires minimal expenditure of personnel time. Our results demonstrated a prevalence of the R2 allele of 11.9% in U.S. Caucasians, 5.6% in African-Americans, 13.4% in Asian or Pacific Islanders and 11.3% in Hispanics. No association between venous thromboembolism and the R2 allele was noted, and furthermore no interaction with FV Leiden was observed.

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Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

Cited by 19 publications

References 3 publications

Poor Response to Activated Protein C as a Prominent Risk Predictor of Advanced Atherosclerosis and Arterial Disease

Poor Response to Activated Protein C as a Prominent Risk Predictor of Advanced Atherosclerosis and Arterial Disease

Double Fluorescent-amplification Refractory Mutation Detection (dF-ARMS) of the Factor V Leiden and Prothrombin Mutations

Factor V Leiden and Factor V R2 Allele: High-throughput Analysis and Association with Venous Thromboembolism

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