four nucleotides, A, C, T and G, enabling the synthesis of roughly 10 15 probes in 4 times 25, or 100 steps. In this way, it’s possible to make an array of virtually any size – including arrays that can examine the entire 3.1 billion bases in the human genome – in 100 steps or less. By reducing array feature size, more probes can be packaged onto the same size surface, increasing the genetic processing power of each individual array. For instance, the first commercial GeneChip products shipped in 1994 had a feature size of 100 microns, that by 2005 have been reduced to 5 microns, allowing 400 times more content on each array. The high data capacity offered by photolithographic manufacturing techniques has provided scientists with tools to study up to 500,000 SNP genotypes per sample analyzed. For any given SNP of two possible genotypes, A or B, probes are synthesized on the array corresponding to both alleles. Following hybridization of the target to the array, scientists can then determine whether a SNP is an AA, AB, or BB genotype by simply analyzing whether the A allele probes have detected their complementary sequence, or whether the B allele probes have detected their complementary sequence, or whether both have detected complementary sequences. 500K: Probe set strategy Every SNP genotype represented on a genotyping microarray is measured through a perfect match probe, as well as a mismatch probe. The mismatch probe serves as an internal control, accounting for spurious signals and cross-hybridization. Each probe-pair is the basic unit used to call a SNP genotype. However, to ensure highly accurate genotype calls, GeneChip arrays routinely use multiple probe pairs to call the genotype for each SNP represented on the array (Fig. 1). The first probe pair contains the SNP precisely in the center of the 25mer sequence. The remaining probe pairs are positioned, or tiled, to the right and the left of this central position. B L I T Z L I C H T This strategy is used to genotype the A and B allele of every SNP from both sense and antisense strands of DNA. The need for high-density manufacturing technique quickly makes itself obvious when genotyping large number of SNPs – more than 10 million probes are used to genotype the 500,000 SNPs represented on the Human Mapping 500K Array Set. 500K: Assay The key to array-based SNP genotyping demanded an assay that would not require allele specific amplifications. And much the way a single assay is used to prepare all transcripts for whole-genome expression analysis, Affymetrix developed a whole genome sampling assay (WGSA) that uses only one primer to genotype hundreds of thousands of SNPs distributed throughout the genome 10 . Previous SNP mapping efforts have been hampered by the need for locus-specific amplification and the need for many tens of thousands of PCR amplifications – an expensive and cumbersome undertaking. The WGSA method uses a simple restriction enzyme to digest genomic DNA, creating various sizes of DNA fragments, each containing their respective SNPs. However, only certain sized fragments are applied to the array, so it’s critical to design microarray probes against those SNPs that are present on the DNA fragments. For example, the Mapping 100K set uses two separate restriction enzyme reactions, each of which creates a pool of DNA fragments containing over 50,000 SNPs to be genotyped. The same strategy is now being used on the 500K to genotype up to 500,000 SNPs – more SNPs than ever before possible. Genotyping up to 10,000 custom SNPs A new generation of genotyping microarrays and assays now enable researchers to perform large-scale genotyping in their own labs with their own panels of SNPs. Highdensity genome-wide genotyping of custom Fig. 1: This microarray probe-set strategy enables scientists to quickly determine whether a genome contains 2 copies of the A allele (AA), two copies of the B allele (BB) or one copy of each (AB). SNPs for focused mapping studies or for candidate-gene association studies requires a method to simultaneously amplify thousands of selected SNPs and an equally scalable way to determine the genotype of each SNP under study. Newly developed MegAllele assays and GeneChip microarrays are now being used to genotype up to 10,000 targeted SNPs using only a single assay and a single microarray. These assays offer researchers the ability to focus on specific regions of the genome more quickly and in greater detail than ever before. Researchers studying candidate genes or other regions of the genome have typically genotyped relatively few SNPs per experiment due to cost and ease-of-use hurdles. The new array-based assay lets scientists successfully genotype more of the SNPs they want in a single, flexible assay. 28 | 6. Jahrgang | Nr. 6/2005 LABORWELT Assay Molecular Inversion Probe (MIP) technology enables scientists to amplify up to 10,000 targeted SNPs in one highly multiplexed experiment that combines 10,000 different PCR reactions in a single tube. When the SNP sequence is amplified during the PCR, a fluorescent base is incorporated at the variable SNP position, and depending on the genotype – either an A, T, C or G – the DNA will contain either a green, blue, purple or red fluorescent molecule. Probe strategy Researchers then use microarrays to detect each SNP sequence, image the associated fluorescent color and ultimately determine the final genotype. Every probe on the microarray surface is designed to detect a different SNP by hybridizing to a DNA sequence that acts as a SNP identifier; each of the 10,000 PCR primers in the initial MIP assay contains one of 10,000 unique DNA sequences that serves as a unique tag for each of the 10,000 single nucleotide polymorphisms. The scalable targeted genotyping method uses a four-color high-resolution scanner to image the fluorescence associated with each probe – red corresponds to a thymine genotype, green to a adenine genotype, blue to a guanine genotype, and purple to a cytosine genotype. If a SNP sequence is imaged as a pure red square, scientists will know that both alleles of the SNP contain a “T” nucleotide – a “T/T” genotype – because only the thymine nucleotides that were incorporated during the MIP assay had red fluorescence.Likewise, if the probe lights up as pure green, scientists know both alleles are an “A” nucleotide – or a “A/A” genotype – because green fluorescence corresponds to adenine. If a probe fluoresces yellow; however, scientists know that the SNP contains one red “T” allele and one green “A” allele that combine to make a yellow “T/A”
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