Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

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21 Citations (Scopus)

Abstract

We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single- tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele- specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the 'in migratio' ethidium-bromide-labeled fragments.

Original languageEnglish
Pages (from-to)816-821
Number of pages6
JournalElectrophoresis
Volume21
Issue number4
DOIs
Publication statusPublished - 2000

Fingerprint

Agar Gel Electrophoresis
Polymerase chain reaction
Electrophoresis
Sepharose
Amplification
Gels
Alleles
Polymerase Chain Reaction
Mutation
Polymorphism
Single Nucleotide Polymorphism
Nucleotides
Ethidium
Factor V
Fluorophores
Reaction products
factor V Leiden
Assays
Screening
Thrombosis

Keywords

  • Factor V Leiden mutation
  • Genotyping
  • Single nucleotide polymorphism

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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title = "Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis",
abstract = "We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single- tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5{\%} of the human population. The rationale of our method is the production of allele- specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the 'in migratio' ethidium-bromide-labeled fragments.",
keywords = "Factor V Leiden mutation, Genotyping, Single nucleotide polymorphism",
author = "M. Sasv{\'a}ri and Arpad Gerstner and Z. R{\'o}nai and M. Staub and A. Guttman",
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T1 - Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

AU - Sasvári, M.

AU - Gerstner, Arpad

AU - Rónai, Z.

AU - Staub, M.

AU - Guttman, A.

PY - 2000

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N2 - We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single- tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele- specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the 'in migratio' ethidium-bromide-labeled fragments.

AB - We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single- tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele- specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the 'in migratio' ethidium-bromide-labeled fragments.

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