Traditional genotyping methods like endonuclease cleavage assays are not suitable for HT genotyping. Even if liquid handling can be optimized, the manual scoring of the cleaved fragments on the gel will remain a major bottleneck. The first major requirement for HT genotyping is therefore the automation of sample preparation until the readout of the genotype. Liquid handling formats used at the moment are 96-well, 384- or 1536-well plates, where genotyping is usually done in large batches.
A second requirement is the availability of sufficient DNA template. Nearly all methods are based on PCR except the invader assay. Although being clearly an elegant assay, it turned out that due to the consumption of DNA in thousands of assays, amounts of source DNA for HT could be prohibitive. Non-PCR methods therefore seem to be a second choice for HT genotyping.
The third criteria for selecting the HT method is the accuracy and the time and effort needed with any given assay. A technician's time for individual optimization of the protocol may be a critical factor. Missing or incorrect genotypes, even in a minor (< 10%) number of samples, may double the time for genotyping. Either individual samples need to be rearrayed in a second step from original plates or—often faster—just repeated from the same source. Average set-up and process time for a single assay is therefore an important point to consider.
The fourth aspect relates to the costs of genotyping. A simple calculation of cent per genotype is of limited value as total costs are caused by buying and maintaining hardware devices for storage, liquid handling, thermocycling, and software for measuring genotypes. Some methods have relative low basic equipment costs of €50,000 (1 euro = $1.2 U.S., March 2006), high set-up costs for single assay (€100-500) with low genotyping costs (€0.20), whereas systems on the opposite site start with basic equipment costs of €500,000, have set-up costs less than €50 but costs per genotype of €0.40. Generally, assays using standard enzymes, nontoxic, nonradioactive reagents will facilitate the laboratory handling and are likely to have even lower prices in the near future.
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