Development of a rapid genotyping method for single nucleotide polymorphisms and its application in cancer studies.
Single nucleotide polymorphisms (SNPs) in cancer-related genes can act as low risk genetic factors for the development of this disease. SNPs have also been shown to influence the efficacy and toxicity of various cytotoxic agents used in the treatment of cancer. Progress in these important areas of cancer research relies upon rapid, inexpensive and accurate means of SNP genotyping. In the present study we describe a fluorescent PCR-based method that utilizes single strand conformation polymorphism (SSCP) analysis to distinguish between different alleles. A real-time DNA fragment analysis platform and ultra-thin, re-useable gels allow short run times and hence a relatively high throughput to be achieved. A standardised procedure for the identification of optimal running conditions for each SNP is presented. We used this fluorescent-SSCP method to genotype SNPs in the MTHFR, p21, cyclin D1, MMP-2, vitamin D receptor, TNF-alpha and IL-6 genes. Multiplex PCR of two SNPs allows up to 500 genotypes per day to be evaluated with 100% accuracy. The low start-up and running costs make this method particularly well suited for SNP genotyping studies that involve up to 1,000 DNA samples.
Grieu
Fabienne
F
Department of Radiation Oncology, Sir Charles Gairdner Hospital, University of Western Australia, Nedlands 6009, Australia.
Joseph
David
D
Norman
Paul
P
Iacopetta
Barry
B
D001483:Base Sequence; D017931:DNA Primers; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational
D017931:DNA Primers; D009363:Neoplasm Proteins