Base Substitutions

Detection of nucleotide changes that don't result in size differences requires other assays for detection. Cleaved amplified polymorphic sequence (CAPS) offer a convenient method of detection when the SNP alters a recognition site for a restriction enzyme (Konieczny and Ausubel 1993). After PCR amplification,^ fragmentsare cutwiththe restrictionenzyme that specifically digests one of the alleles. The digestion fragments are then separated on an agarose gel and scored. However, CAPS are limited to mutations that create or disrupt a restriction enzyme recognition site. When no recognition site is detected an alternative to CAPS is derived cleaved amplified polymorphic sequence (dCAPS), where PCR primer mismatches are used to create a polymorphism based on the target mutation (Neff et al. 1998). The program dCAPS Finder 2.0 (Table 5) facilitates the design of these PCR primers. Both CAPS and dCAPS markers are reliable and easy to apply. However, throughput is limited due to agarose gel electrophoresis. Furthermore, the analysis requires the use of endonucleases which are often expensive, thus limiting the wide-spread use of CAPS and dCAPS in molecular mapping.

Other SNP detection platforms discriminate between alleles using allele-specific primer extension (ASPE) assays. Allele discrimination is accomplished through a variety of detection systems and platforms (Kwok 2001). One of these platforms is offered by Lu-minex (http://www.luminexcorp.com), and uses the principles of flow cytometry to detect polymorphisms using ASPE. Two allele-specific primers are designed for each SNP, with the 3' base specific to the polymorphism and a tag attached to the 5' end. Successful extension with a biotin labeled base of the perfect matching primer allows products to be enriched and sorted using beads. Laser detection identifies the micro-bead and quantifies the signal. With 100 distinct sets of micro-beads, up to 50 bi-allelic SNPs can be assayed in a single reaction (Lee et al. 2004b) resulting in approximately 4,000 data points per day. Another procedure designed specifically for high-throughput SNP detection is offered by the Illumina Golden Gate Assay (http://www.illumina.com). In this procedure, hundreds of SNP-specific oligonucleotides are simultaneously hybridized directly to genomic DNA. These oligonucleotides contain target sequences for a set of universal primers and address sequences recognized by labeled beads in the array. Throughput is very high, although this system is only cost-effective when analyzing large datasets.

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