Table 6.

Methods of allelic discrimination used in SNP genotyping [63].

Enzymatic cleavage	Enzymatic cleavage is based on the ability of certain classes of enzymes to cleave DNA by recognition of specific sequences and structures. Such enzymes can be used for discrimination between alleles when SNP sites are located in an enzyme recognition sequence and allelic differences affect recognition. For example, restriction fragment length polymorphism (RFLP) is based on genotyping a SNP located in a restriction enzyme site using PCR product containing the SNP that is incubated with corresponding restriction enzyme. The reaction product is run on a gel, and SNP genotype is easily determined from the product sizes [64].
Primer extension	In a typical primer extension reaction, a primer is designed to anneal with its 3\ end adjacent to a SNP site and extended with nucleotides by polymerase enzyme. The identity of the extended base is determined either by fluorescence or mass to reveal SNP genotype, for example, the PinPoint assay, MassEXTEND tm, SPC-SBE, and GOODassay primer extension-based methods, where SNP-specific primers are simultaneously extended with various nucleotides using PCR products as a template [65].
Hybridization	Hybridization approaches use differences in the thermal stability of double-stranded DNA to distinguish between perfectly matched and mismatched target-probe. For example, the TaqMan® genotyping assay combines hybridization and 5\ nuclease activity of polymerase coupled with fluorescence detection. The allele-specific probes carry a fluorescent dye at one end (reporter) and a nonfluorescent dye at the other end (quencher). The intact probes show no fluorescence owing to the close proximity between the reporter and quencher dyes. During PCR primer extension, the enzyme only cleaves the hybridized probe that is perfectly matched, freeing the reporter dye from the quencher. The reporter dye generates a fluorescent signal, whereas the mismatched probe remains intact and shows no fluorescence [66].
Ligation	Ligation approach employs specificity of ligase enzymes. When two oligonucleotides hybridize to single-stranded template DNA with perfect complementarity, adjacent to each other, ligase enzymes join them to form a single oligonucleotide. Three oligonucleotide probes are used in traditional ligation assays, 2 of which are allele-specific and bind to the template at the SNP site. The third probe is common and binds to the template adjacent to the SNP immediately next to the allele-specific probe. For example, combinatorial fluorescence energy transfer tags are composed of fluorescent dyes that can transfer energy when they are in close proximity. Tags with different fluorescence signatures can be created using a limited number of dyes by varying the number of dyes used and spacing between the dyes [67].