SBE-TAGS: An array-based method for efficient single-nucleotide polymorphism genotyping

Abstract

Generating human single-nucleotide polymorphisms (SNPs) is no longer a rate-limiting step for genetic studies of disease. The number of SNPs in public databases already exceeds 200,000, and the total is expected to exceed 1,000,000 within a year. Rather, progress is limited by the inability to genotype large numbers of SNPs. Current genotyping methods are suitable for studying individual loci or at most a handful at a time. Here, we describe a method for parallel genotyping of SNPs, called single base extension-tag array on glass slides, SBE-TAGS. The principle is as follows. SNPs are genotyped by single base extension (SBE), using bifunctional primers carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the genotyping reactions can be performed in a highly multiplexed fashion, and the resulting product can then be ''demultiplexed'' by hybridization to the reverse complements of the sequence tags arrayed on a glass slide. SBE-TAGS is simple and inexpensive because of the high degree of multiplexing and the use of an easily generated, generic tag array. The method is also highly accurate: we genotyped over 100 SNPs, obtaining over 5,000 genotypes, with approximately 99% accuracy.

A review on SNP and other types of molecular markers and their use in animal genetics

“…Multiplexing of this procedure has been described thus reducing costs and improving throughput. In these methods, the different loci genotyped simultaneously are separated either by gel electrophoresis [49] or by hybridisation to arrayed tags [32]. Recently, Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) was developed as a tool for differentiating genotypes, by comparing the mass of DNA fragments after a single ddNTP primer extension reaction, in which no labelling is necessary.…”

Development of a routine procedure for single nucleotide polymorphism marker design based on the Tm-shift genotyping method

“…A number of techniques for SNP genotyping have been reported, including those based on allele-specific PCR (Sommer et al 1992), single-base extension (Syvanen 2001), probe hybridization (Livak et al 1995, Piatek et al 1998, Schutz et al 2000, and microarray technology (Hirschhorn et al 2000), among others (Kwok 2001). Each method has advantages and disadvantages compared with the others in regard to cost, specificity, and throughput, and no clearly superior method has been established.…”

CYP2D6 Genotyping with Oligonucleotide Microarrays and Nortriptyline Concentrations in Geriatric Depression

“…Microarrays of far higher density are feasible using currently available technology. A "custom" array designed to identify polymorphisms in other candidate pharmacogenes for antidepressant side effects and/or efficacy could be devised and implemented immediately using a new generic microarray format (Hirschhorn et al 2000). This could expedite the screening of large numbers of subjects treated with antidepressants for genetic determinants of medication efficacy and tolerance.…”