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Accessing genetic variation: genotyping single nucleotide polymorphisms

Key Points

  • There are intense continuing efforts to increase the throughput and accuracy, and reduce the costs, of methods for genotyping single nucleotide polymorphisms (SNPs). This is driven by the hope that SNPs can act as markers for identifying the genes that underlie multifactorial disorders.

  • PCR, invented in the 1980s, allows the sensitivity and specificity required for genotyping SNPs in large diploid genomes. The PCR step is the principal limiting factor in the throughput of current SNP-genotyping assays.

  • The large number of different SNP assays are based on a small number of reaction principles that have been combined with solid-phase or solution-based assay formats. Fluorescence is the most frequently used detection method.

  • Allele-specific oligonucleotides as probes or PCR primers are used to achieve high throughput in homogeneous solution phase assays that are monitored in real time during PCR. Alternatively, large numbers of oligonucleotide probes are immobilized at high density on microarrays to allow parallel analysis of many SNPs.

  • The most promising methods for accurate genotyping of SNPs involve nucleic-acid-modifying enzymes as genotyping tools. Frequently used enzymes are DNA polymerases, ligases and endonucleases.

  • Assays based on primer extension catalysed by a DNA polymerase are robust and have been adapted to various assay formats and detection strategies. These include colorimetric detection in microtitre plates, fluorescence detection using DNA sequencers, mass spectrometric detection and microarray-based assays with fluorescence detection.

  • Assays based on DNA ligation, or cleavage by FLAP endonucleases, have led to the development of SNP-genotyping methods in which a PCR amplification step is avoided. Instead, an enzymatic signal amplification scheme is used to obtain sufficient sensitivity.

  • Future SNP assays could be based on PCR carried out in microcapillaries streamlined with one of the enzymatic detection principles, and the assays could be multiplexed by combinatorial fluorescent labels.


Understanding the relationship between genetic variation and biological function on a genomic scale is expected to provide fundamental new insights into the biology, evolution and pathophysiology of humans and other species. The hope that single nucleotide polymorphisms (SNPs) will allow genes that underlie complex disease to be identified, together with progress in identifying large sets of SNPs, are the driving forces behind intense efforts to establish the technology for large-scale analysis of SNPs. New genotyping methods that are high throughput, accurate and cheap are urgently needed for gaining full access to the abundant genetic variation of organisms.

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Figure 1: 'Modular' design of some of the assays for SNP genotyping.
Figure 2: SNP genotyping by minsequencing using an 'array of arrays'.


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The development and application of technology for single nucleotide polymorphism genotyping in my laboratory is supported by the Swedish Research Council and by the Wallenberg Consortium Nord. I thank Å. Dahllöf and A. Bernsel for help with the reference list.

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apolipoprotein E

factor V Leiden


Affymetrix (GeneFlex®, GeneChips)

AP Biotech (rolling circle amplification)

Applied Biosystems (TaqMan, SnaPshot, PinPoint assay)

Celera Genomics

Encyclopedia of life sciences

Complex multifactorial genetic diseases

Genetic variation: human



Molecular Beacon

Molecular Staging (rolling circle amplification)

Orchid Biosciences (Genetic Bit Analysis, SNPstream)

Packard Bioscience (AlphaScreen)

Perkin Elmer Life Sciences (TDI)

Pyrosequencing AB

Sequenom (MassArray)

SNP Consortium

ThermoHybaid (DASH)

Third Wave Technologies (Invader assay)



Analysing single nucleotide polymorphism alleles in population-based studies to identify loci that are associated with a particular disease or phenotype.


A genotyping method based on hybridization between allele-specific oligonucleotide probes that have been immobilized on a membrane, and amplified DNA fragments in solution.


(PNA). Biopolymer molecule that consists of DNA bases connected by a backbone of peptide bonds instead of phosphodiester bonds as in natural DNA.


(LNA). DNA analogues in which the 2′ and 4′ positions in a furanose ring are connected by a methylene moiety.


A phenomenon by which the energy from an excited fluorophore is transferred to an acceptor molecule at short (<100 Å) distances, leading to quenching of the fluorescence. The efficiency of energy transfer depends strongly on the distance between the donor and acceptor molecules.


Unwanted PCR products formed when two primers interact during the extension phase of PCR, followed by extension of the 3′-end of one or both primers with the other primer acting as a template.


(enzyme-linked immunosorbent assay). A widely used immunochemical method for detecting antigens or antibodies. ELISA methods are carried out in microtitre plates and use colorimetric detection.


Small molecule that is able to invoke an antibody response when used for immunization of an animal.


A method for DNA sequencing, in which the inorganic pyrophosphate (PPi) that is released from a nucleoside triphosphate on DNA chain elongation is detected by a bioluminometric assay.


(also known as microparticles or microbeads). Small 1–100-μm diameter particles used as solid supports in bioassays. They can carry a probe or primer, and can contain internal magnetic compounds to allow magnetic separation or internal fluorescent compounds for labelling.


A detection method based on excitation of a fluorescent molecule by plane-polarized light, and measurement of the rate of depolarization of fluorescence. This rate is proportional to the rate of tumbling of a fluorescent molecule. As small molecules tumble faster than large molecules in solution, fluorescent molecules of different sizes can be distinguished.


A detection system in which excitation of fluorophores takes place only in a small three-dimensional focal volume.


Nanocrystal that consists of a core of cadmium selenide wrapped with multiple monolayers of zinc sulphide that have several times higher extinction coefficients than organic fluorophores. The quantum dots can be excited with light of a single wavelength, and emit very bright fluorescence at several wavelengths that are determined by the size of the cadmium selenide core.

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Syvänen, AC. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet 2, 930–942 (2001).

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