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Direct Sequencing Is More Accurate and Feasible in Detecting Single Nucleotide Polymorphisms than RFLP: Using Human Vascular Endothelial Growth Factor Gene as a Model

Center for Injury Mechanisms and Related Responses, Gene Therapy Group, at the University of Arizona, Tucson, ahtdavis{at}nursing.arizona.edu

Jianhua Wang, MS

Center for Injury Mechanisms and Related Responses, Gene Therapy Group, at the University of Arizona, Tucson

Tom C. Tsang, PhD

Department of Microbiology & Immunology, Gene Therapy Group, at the University of Arizona, Tucson

David T. Harris, PhD

Department of Microbiology & Immunology, Gene Therapy Group, at the University of Arizona, Tucson

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.

Key Words: gene • single nucleotide polymorphism • vascular endothelial growth factor • restriction fragment length polymorphism • direct sequencing • methodology • accuracy

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