High resolution SNP detection methods

High resolution SNP identification relies on sequence-based typing (SBT), which can recognize known SNPs in homo- or heterozygous conditions and spot unknowns. Capillary sequencing makes this method semi-high throughput. However, the majority of SNPs are dispersed across the whole genome, with few exceptions such as the HLA region, at an average 1-kb distance from each other. Since accurate sequencing is limited to about 800-1000 bp per sequencing reaction, SBT is not an efficient method for high throughput sequencing of most genomic regions. Furthermore, the cost of SBT is not affordable for most research facilities. Finally, even sequencing has its own limitations since it does not allow segregating cis-to trans- ambiguities in heterozygous conditions when more than one SNP is detected in the region being sequenced (14). Pyrosequencing is a newly developed real time quantitative sequencing method that may complement high-throughput SBT since it can resolve cis/trans ambiguities. Using a programmed nucleotide dispensation order (NDO), pyrosequencing is especially suitable for the detection of SNPs when their frequency is low and for studies using pooled samples (15, 16). The drawback of this method is its cost, the complexity of designing NDOs and the special equipment and reagents necessary.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful tool for genotyping SNPs (17-19). The comprehensive utilization of this approach is, however, hampered by the complexity of the technology, the cost and the limited throughput.

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