Factor V Leiden Mutation Screened by PCR and Detected with Lanthanide-Labeled Probes

Potter, Christine; Liu, Y T; Rees, David C.

doi:10.1089/109065701753617417

Cited by 6 publications

(5 citation statements)

References 52 publications

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“…Approximately 5% of Caucasians are either heterozygous carriers or homozygous for the factor V Leiden mutation; the prevalence is reported to be lower for other ethnic groups . Potter et al have developed a simplified and robust assay using oligonucleotide probes for normal and mutant factor V Leiden mutation sequences, labeled with europium and samarium, respectively, and measured by time-resolved fluorescence. The electronic array analysis of amplicons for factor V mutation by using strand displacement amplification was described. − After electronic addressing of the amplicons to specific array locations, the reporter oligonucleotides modified with either Cy3 or Cy5 were added, and fluorescence at each location was quantified.…”

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confidence: 99%

Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor

et al. 2002

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An electrochemical genosensor for the genotype detection of allele-specific factor V Leiden mutation from PCR amplicons using the intrinsic guanine signal is described. The biosensor relies on the immobilization of the 21-mer inosine-substituted oligonucleotide capture probes related to the wild-type or mutant-type amplicons, and these probes are hybridized with their complementary DNA sequences at a carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The guanine signal was monitored as a result of the specific hybridization between the probe and amplicon at the CPE surface. No label-binding step was necessary, and the appearance of the guanine signal shortened the assay time and simplified the detection of the factor V Leiden mutation from polymerase chain reaction (PCR)-amplified amplicons. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the guanine signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 51.14 fmol/mL target DNA. With the help of the appearance of the guanine signal, the yes/no system is established for the electrochemical detection of allele-specific mutation on factor V for the first time. Features of this protocol are discussed and optimized.

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Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor

et al. 2002

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“…Studies varied greatly in the extent to which they described the population from which the participants came; only 17 studies adequately reported the setting or population from which the blood samples were obtained [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Most described the experimental test adequately; 60 to 70% of the studies gave a detailed description of their reference methods.…”

Section: Resultsmentioning

confidence: 99%

“…Three other studies used a fluorophore probe to detect FVL [46][47][48], and all reported 100% concordance. Potter et al tested a lanthanide-labeled probe for FVL detection [19]. There was 1% discordance on the first test, and 0.26% remained discordant after repeating the test.…”

Section: Detection Of Fvlmentioning

confidence: 99%

Analytic validity of genetic tests to identify factor V Leiden and prothrombin G20210A

et al. 2010

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The objective of this study is to systematically review methods for detecting Factor V Leiden or prothrombin G20210A. English-language literature from MEDLINE 1 , EMBASE 1 , The Cochrane Library, the Cumulative Index to Nursing and Allied Health Literature, PsycInfo , 2000-December 2008. Studies assessed methods for detection of these mutations in at least 10 human blood samples and reported concordance, discordance, or reproducibility. Two investigators abstracted data on the sample selection criteria, test operators, DNA extraction, experimental test, reference standard, commercial instruments, concordance rates, explanation of any discordance, and whether discordance resolved after repetition. We assessed strength of the evidence using the GRADE criteria. We reviewed 7,777 titles and included 66 articles. The majority of the reviewed studies used PCR-RFLP or AS-PCR as the reference standard. The studies demonstrated that commercially available and precommercial tests have high analytic validity with all having greater than 99% concordance with the reference standard. With a few exceptions, discordance resolved with repetition of the test, suggesting operator or administrative errors were responsible for the discordant results. In the quality assurance studies, greater than 98% of laboratories demonstrated high, even perfect, accuracy when asked to diagnose a sample with a known mutation. The majority of errors came from a limited number of laboratories. Although not all methods may be accurate, there is high-grade evidence that genetic tests for the detection of FVL and prothrombin G20210A have excellent analytic validity. There is high-grade evidence that most, but not all, clinical laboratories test for FVL and prothrombin G20210A accurately. Am. J. Hematol. 85:264-270, 2010. V

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“…Genetic diagnosis of factor V Leiden based on a heteroduplex technology (14) and PCR based methods with variously labeled primers combined with different detection/separation techniques have also been described (15,16). Even though heteroduplex-based mutation detection comprises a single PCR reaction followed by PCR product analysis, it uses either standard or capillary polyacrylamide gel electrophoresis (PAGE).…”

Section: Discussionmentioning

confidence: 99%

Detection of Factor V Leiden by PCR-SSCP using GMATM Precast Elchrom Scientific Gels

Šimundić¹,

Topić²,

Štefanović³

2003

Clin Appl Thromb Hemost

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Genetic abnormalities in hemostatic proteins associated with hypercoagulability are an important hereditary risk factor for venous thrombosis. Several genetic mutations that cause hereditary disorders predisposing to thrombosis have been described, point mutation in the coagulation factor V gene (FV:R506Q), called factor V Leiden, being the most common of them. A new inexpensive and simple polymerase chain reaction-single-strand polymorphism (PCR-SSCP) based method for detection of this genetic abnormality is reported. The study population consisted of 150 subjects whose factor V genotype was previously determined by PCR-RFLP method using the Mnl I restriction endonuclease. A 223-bp fragment containing the G1692-A (Arg 506-Gln) polymorphic site in exon 10 of the factor V gene was amplified, denatured, and run overnight on the commercially available GMA gels for SSCP. PCR-SSCP analysis showed reproducible and uniform band patterns for FV mutant and wild type alleles. Furthermore, PCR-SSCP results were consistent with those obtained with PCR-RFLP analysis (100%). The described PCR-SSCP procedure is reliable, time-saving, and cost-effective. The method may be considered as a potentially powerful new tool in the routine detection of factor V Leiden.

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Factor V Leiden Mutation Screened by PCR and Detected with Lanthanide-Labeled Probes

Cited by 6 publications

References 52 publications

Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor

Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor

Analytic validity of genetic tests to identify factor V Leiden and prothrombin G20210A

Detection of Factor V Leiden by PCR-SSCP using GMATM Precast Elchrom Scientific Gels

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