Direct sequencing is more accurate and feasible in detecting single nucleotide polymorphisms than RFLP: Using human vascular endothelial growth factor gene as a model

Amy H.T. Davis, Jianhua Wang, Tom C. Tsang, David T. Harris

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Since the sequencing of the human genome, there has been increased interest in understanding the distribution and effects of genetic variations among individuals. Restriction fragment length polymorphism (RFLP) is a well-established and frequently used method for genotyping. This method, however, is indirect and has a number of limitations. It is thus important to reevaluate the use of RFLP in light of more contemporary methods of genotyping. The specific aims of this study are to (a) compare genotyping methods of traditional RFLP with contemporary direct sequencing for accurate identification of polymorphisms within the human vascular endothelial growth factor (VEGF) gene and (b) describe distribution of a known single nucleotide polymorphism (SNP) in the VEGF gene in a sample composed of 50 healthy volunteers. Polymerase chain reaction (PCR) was used to amplify the initial sample of DNA. Genotypes of a G-to-A substitution (GG, AG, AA) at -1154 were analyzed by RFLP and direct sequencing. RFLP was unable to discriminate among the three possible genotypes, whereas direct sequencing clearly identified genotype for all 50 samples. Observed genotype frequencies were comparable with the Hardy-Weinberg principle. This comparative study provides justification for selecting direct sequencing instead of RFLP for detecting SNPs in selected genes.

Original languageEnglish (US)
Pages (from-to)170-178
Number of pages9
JournalBiological Research For Nursing
Volume9
Issue number2
DOIs
StatePublished - Oct 1 2007

Keywords

  • Accuracy
  • Direct sequencing
  • Gene
  • Methodology
  • Restriction fragment length polymorphism
  • Single nucleotide polymorphism
  • Vascular endothelial growth factor

ASJC Scopus subject areas

  • Research and Theory

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