Current methods (Table 1, Fig. 1) combine at least one of the four different principles of allelic discrimination (hybridization, primer extension, ligation, or restriction) with one of the four different detection techniques (chemoluminescence, fluorescence polarization, resonance energy transfer, and mass spectrometry). Assay formats range from (slab-)gel electrophoresis, plates, particles, fiber arrays and microchip arrays to semi- and homogenous assays that do not require any further sample separation or purification. Whereas homogeneous assays are flexible and probably not labor-intensive, the amount of multiplexing is usually limited. Solid-phase reactions can be carried out on glass slides, silicon chips, and magnetic beads. In other assay designs, defined oligonucleotides are attached to the solid phase, and samples interrogate the alleles being already printed on a chip. Discrimination in such assays is highly parallel and saves the time and efforts for setting up multiple individual reactions. A major drawback is that the custom design is not very flexible and can be done by the vendor only.
The different detection methods also have marked differences. Numerous labels are available, most of them with light-emitting properties that enable elegant assays. When the reaction product is separated from the initial reaction, usually only one label is necessary. Homogenous assays otherwise require two labels that change their property during the reaction. Fluorescence detection methods have higher set-up costs by the synthesis of
Table 1 High-Throughput Genotyping Methods
Microarrays 5' exonuclease assay, allele-specific hybridization, real-time PCR
Molecular beacons Dynamic allele-specific hybridization Fiberoptic analysis of coded tags Minisequencing
Hapten-labeled nucleotides Chemoluminometric detection of pyrophosphate MALDI-TOF mass spectrometry Allele-specific PCR,
FRET energy transfer Colorimetric OLA Ligation and fiberoptic detection Upstream inserted oligonucleotide cleaved by flap endonuclease
Highly parallel, fast Simple assay
Versatile assay Inexpensive labeling
Highly parallel, fast
Additional sequence information
Fast set-up, accurate
Highly parallel No PCR necessary
GeneChipw (Affymetrix) TaqManw (Applied Biosystems)
Hybaid) Sentrex® (Illumina)
Biosystems) SNPit® (Orchid)
Mass Extendw (Sequenom) Alpha Screen® (Packard Biosystems)
Illuminaw (Bead Array)
Invader® (Third Wave)
Abbreviations: MALDI-TOF, matrix-assisted laser desorption/ionization time-of-light; PCR, polymerase chain reaction; FRET, fluorescence resonance energy transfer; OLA, oligonucleotide ligation assay; DASH, dynamic allele specific hybridization.
specific labeled primer, whereas no modified primers are necessary for mass spectrometry measurements. Although being the most direct and accurate approach, mass detection still has the disadvantage of a relatively expensive and a technically demanding equipment.
All systems are scalable from a few to many thousand genotypes per day. The limiting factor for HT genotyping probably lies more in general sample preparation than scoring of the genotypes. Typing more than 100,000 genotypes per day may even generate bottlenecks of the analysis and interpretation of the generated data.
The need of software organizing HT by assay and sample tracking is often underestimated. Thorough monitoring of the movement of all plates at all positions is critical for quality control in a HT genotyping laboratory. Most genotyping groups are using proprietary databases to manage this task. Usually client computer on freezer, liquid handlers, thermocyclers, and analysis devices are connected to a main database that monitors in-time the process flow. A single sample is identified by the address on a plate grid, which is linked back to the original sample. During assay set-up, every plate is linked to a particular assay, which by itself links to a genotype, making it possible to assign a genotype to an individual study participant.
From many assays available at the moment, we will now discuss in detail five methods that are most widely used. Additional methods may be found elsewhere (1-3).
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