Single nucleotide polymorphisms (SNPs) are single base pair mutations at specific locations in the coding and non-coding regions of the genome that are found in more than 1% of a population[1]. While SNPs located within a coding region are most likely to alter the biological function of a protein, SNPs found within the non-coding regions may affect gene regulation. Both, coding and non-coding SNPs have been associated with population-specific disease development or drug susceptibility and are therefore promising molecular markers for disease genetics and pharmacogenomics studies[2].
An efficient and reliable approach for genotyping of a priori known SNP locations is single basepair extension (SBE)[3,4,5]. This method relies on the extension of a primer, designed to bind one nucleotide upstream of the polymorphic spot, by a SNP-complementary, fluorescently labeled ddNTP[5]. Subsequent detection of the incorporated ddNTP is performed by fluorescent visualization of the extended primer by electrophoresis or array-based methods, thus revealing the nucleotide base at that position on the template strand.
Our fluorescently labeled ddNTPs are > 95 % pure (HPLC) and have been successfully used in SBE experiments[5]. Further fluorescently labeled ddATP, ddCTP, ddGTP and ddUTP analogs (e.g. Cy3 or Cy5-labeled), amine-labeled ddNTPs for subsequent coupling of NHS-ester-modified fluorescent dyes as well as biotin-labeled ddNTPs for subsequent detection with fluorescent streptavidin are available as catalog products.
Figure 1: Single nucleotide polymorphisms (SNPs) are detected by enzymatic elongation of a primer by a SNP-complementary, fluorescently labeled ddNTP. The primer is designed to anneal one base upstream of the SNP site. Subsequent fluorescent visualization of the product reveals the incorporated nucleotide and hence the SNP genotype.
[1] Brookes et al. (1999) The essence of SNPs. Gene 234:177.
[2] Kim et al. (2007) SNP genotyping: technologies and biomedical applications. Annual review of Biomedical Engineering 9:289.
[3] Desphande et al. (2005) Multiplexed SNP Genotyping using single-base extension (SBE) and microsphere arrays. Current protocols in Cytometry 13 (4):1.
[4] Syvänen et al. (1999) From gels to chips: Minisequencing primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mut. 13:10.
[5] Esteves et al. (2011) Clinical relevance of multiple single-nucleotide polymorphisms in Pneumocystis jirovecii Pneumonia: development of a multiplex PCR-single-base-extension methodology. J. Clin. Microbiol. 49 (5):1810.
