The SNPlex Genotyping System consists of a set of pre-optimized, universal assay reagents that are utilized independently of the genotypes studied. The only SNP-specific components of the assay are the ligation probes that participate in the oligonucleotide ligation (OLA). Currently, up to 48 SNPs can be addressed simultaneously in one OLA reaction.
The assay workflow for the SNPlex Genotyping System involves the following seven steps, designed for easy automation, which can be completed within two days (Figure 1): (1) allele-specific OLA reaction; (2) purification of OLA reaction by exonucleolytic digestion of excess probes and linkers; (3) universal PCR reaction to amplify ligation products; (4) capturing of biotin-labeled PCR products in streptavidin coated microtiter plates; (5) binding of ZipChute probes to single-strand PCR products; (6) elution of hybridized ZipChute probes; and (7) detection by CE.
FIGURE 1 A SNPlex Genotyping System assay protocol can be completed within two days. On the first day, the OLA reaction, exonuclease purification, and PCR amplification are performed. On the second day, the amplicons are immobilized on streptavidin-coated microtiter (more ...)
Step one consists of the OLA reaction, during which allele-specific oligonucleotide (ASO) probes and locus-specific oligonucleotide (LSO) probes hybridize to the genomic target sequence. Typically, 37 ng of gDNA is used, resulting in the consumption of <1 ng of gDNA per genotype. These allele-specific and locus-specific probes ligate when they are hybridized to a perfectly matching sequence at the SNP site. Simultaneously, universal linkers are ligated to the distal termini of the ASO and LSO ligation probes. These linkers contain universal PCR primer–binding sequences as well as sequences complementary to ASO and LSO probes. A unique ZipCode sequence is attached at the 5′ end of the genomic equivalent sequence within each ASO. Consequently, by virtue of the ZipCode sequence, the OLA step encodes the genotype information of every SNP into unique ligation products. All probes are designed to function under the same hybridization conditions; therefore, no optimization of OLA reaction conditions is required.
In step two, unligated probes and linkers, as well as the genomic DNA, are removed by enzymatic digestion using exonuclease I and lambda exonuclease. This step is necessary to ensure the efficiency of the subsequent PCR reaction. Step three involves the simultaneous PCR amplification of purified ligation products with a single pair of PCR primers, one of which is biotinylated. Since we use a universal pair of PCR primers, no optimization of PCR reaction conditions is necessary. Next (step four), biotinylated amplicons are bound within wells of streptavidin-coated microtiter plates. Subsequently, the non-biotinylated strands are removed, leaving single-stranded amplicons bound to the microtiter plate.
In step five, fluorescently labeled universal Zip-Chute probes hybridize to the bound single-stranded amplicons. Each ZipChute probe contains a sequence complementary to the unique ZipCode sequence within each ASO; therefore, in order to analyze 48 SNPs, 96 different ZipCode sequences and 96 unique ZipChute probes are required. Each ZipChute probe further contains a mobility modifier, which assigns to each ZipChute probe a specific rate of mobility during CE. Finally, in steps six and seven, the specifically bound ZipChute probes are eluted into CE buffer and analyzed on an Applied Biosystems 3730/3730xl DNA Analyzer.
GeneMapper software is used for analyzing the raw CE data and calling SNP genotypes (Figure 2). Because one SNP is typically characterized by two possible alleles, two fluorescent peaks in a CE electropherogram represent the two alleles of a specific SNP. GeneMapper analysis software assigns individual genotypes, based on the intensity and location of peaks.
FIGURE 2 GeneMapper software analyzes the electropherogram of a SNPlex system sample (A). Two neighboring peaks indicate the genotype of a particular SNP. The software creates for each SNP a genotype plot and uses a clustering algorithm to assign genotypes. SNP (more ...)
In this paper we describe the performance of the SNPlex Genotyping System. We used a probe set, called the control pool, which interrogates 48 population-validated SNPs (Table 1). We tested this control pool against 44 genomic DNAs, which were each represented eight times on a 384-well plate. In addition, we describe the design and performance of 11 probe sets that analyzed 521 SNPs in 92 individuals. For both studies, we report the pass rate, call rate, precision, and concordance with data from TaqMan probe-based assays.