Spurred on by the need to type thousands of SNPs (Single Nucleotide Polymorphisms) for human disease association studies, a variety of technologies have been developed for SNP analysis.  Most methods are amplification based and the SNP is subsequently detected by primer extension, oligonucleotide ligation, or hybridization of a probe to the amplified product (see figure below).  In some ways, the best SNP-typing system is that of standard DNA Sanger sequencing, in which the SNP site and flanking sequences are simultaneously interrogated.  However, such an approach is limited to densely packed SNPs, such as those present in the mtDNA control region.  For the other SNP applications in which the SNP sites are separated by thousands (often hundreds of thousands) of bases, Sanger sequencing is neither practical nor cost effective.

The SNP analysis technology that has been used most productively for the coding region mtDNA, physical characteristics, and degraded DNA applications is based upon primer extension (or mini-sequencing) followed by capillary electrophoresis (CE) or solid phase capture.⁰⁹ The SNaP shot™ assay (AB), which has been used for the mtDNA coding region analysis, uses a primer that abuts the SNP site, genomic DNA template, DNA polymerase, and fluorescently labeled terminator ddNTPs. The SNP site on the genomic template will determine, by complementary Watson-Crick base pairing, which ddNTP will be extended and incorporated into the primer. The now fluorescently labeled extended primer is separated by capillary electrophoresis, and the allelic status determined by the nature of the fluorescent dye detected. Multiplexing is achieved by designing primers of different lengths (or containing mobility modifiers) at the primers' 5' ends, such that each separate SNP in the multiplex has a characteristic mobility.

Read more on the SNap shot™ assay on Applied Biosystems website.

The SNPstream^® UHT (Orchid Cellmark) is a primer extension methodology that has been used to predict bio-geographical ancestry and to analyze degraded DNA. This platform uses SNP extension primers that are modified at the 5' ends with oligonucleotide tags that are complementary to oligonucleotides that are bound to a solid microplate support.  Each well of the microplate has 16 anti-tag sequences. Hybridization to it of a SNP primer extension reaction, previously carried out in solution, permits multiplex typing by location (determines the SNP locus) and bound dye (determines the allelic status).

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