SNPWave® is a novel multiplexed technology, capable of detecting various subsets of sequences, such as single nucleotide polymorphisms (SNPs), in a flexible fashion. The SNPWave technology targets the market segment defined by moderate numbers of SNPs (up to several hundreds) and medium to high number of samples. This includes both (human) diagnostic analyses and agricultural applications such as genetic mapping, genetic diversity analysis and marker-assisted breeding, in a wide variety of species including plants, mammals, and micro-organisms. With respect to target sequences, applications of the SNPWave technology are not limited to SNPs per se, but may also include detection of non-polymorphic sequences (introgression segments, transgenes, pathogens), detection of low-abundant sequences in a complex background and/or selected combinations of transcripts for diagnostic prediction of complex traits.
HOW IT WORKS
SNPWave is based on highly multiplexed ligation, followed by selective amplification of 20 -60 ligated probes in a single polymerase chain reaction using the AFLP® technology (Figure 1). Detection of SNPWave reaction products is based on size separation on a sequencing instrument with multiple fluorescence labels and short run times. By combining multiplexed ligation-dependent amplification with the known robustness of AFLP, low-cost probe synthesis, and high throughput detection using flanking sizing standards, SNPWave allows detection of SNPs under uniform reaction conditions in a highly flexible way. At present, 138,240 SNPs can be scored within 24 hours on a single MegaBACE 1000 with 96 capillaries, using 10-plex amplifications, four fluorescent dyes and 36 short runs with a flanking sizing standard. In addition to length-based detection, the SNPWave technology can also be combined with hybridization- and mass-based detection platforms. The power, robustness and flexibility of the SNPWave technology is illustrated by a 100-plex assay for SNP genotyping in Arabidopsis.
The SNPWave technology is well suited for high throughput analysis of thousands of samples and is easily transferred to laboratories familiar with PCR work. At this moment KeyGene develops custom 10-90-plex assays for in-house use by its customers.
Figure 1. Principle of the SNPWave method. Allele-specific ligation probes are hybridized to denatured genomic DNA. SNP allele discrimination is based on the specificity of the Taq (Thermus Aquaticus) DNA ligase. (A/B) Closed circular probes are formed only in case the 3’ hydroxylated SNP allele-specific end of the ligation probe hybridizes immediately adjacent to the 5’ phosphorylated common probe sequence of the opposite end of the probe. (C) Next, closed circular probes are amplified, commonly with AFLP primers containing two selective nucleotides.
Figure 2. Genotyping of 69 Arabidopsis accessions using the SNPWave technology.
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