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Clin Biochem Rev. 2006 February; 27(1): 63–75.
PMCID: PMC1390795

Genotyping of Single Nucleotide Substitutions

Abstract

The description of the polymerase chain reaction in 1985 caused a revolution in genetics and today molecular diagnostics is one of the leading growth areas across all disciplines of laboratory medicine. This paper reviews the principles and limitations of a number of traditional and emerging techniques for typing of single nucleotide substitutions. The techniques discussed include traditional approaches such as restriction enzyme analysis, more recent homogenous methods, such as those utilising TaqMan®, fluorescence resonance energy transfer (FRET) and Scorpion® probes, and high resolution melting curve analysis. Non-homogenous but highly flexible approaches such as Pyrosequencing™ and mass-spectrometry are also discussed. While many techniques are available, it is clear that no one approach is clearly superior. However, in terms of their many advantages and continuing developments, homogenous approaches have much to recommend them.

Introduction

Variation in the human genome is characterised by numerous types of mutations and polymorphisms, including insertions and deletions, the expansion of tandem repeat sequences, and single nucleotide substitutions. The latter are also known as single nucleotide polymorphisms or SNPs when they are relatively common, and more specifically, when the frequency of the least frequent variant, or minor allele, exceeds 1%. Single nucleotide substitutions are considered to be by far the most common type of variation and occur at a frequency of approximately one in every 1000 nucleotides and world-wide, public efforts have so far identified over 7 million common SNPs. These are finding increased use in fine mapping of simple genetic disorders, in the delineation of genetic influences in polygenic or multifactorial diseases such as stroke, coronary heart disease and asthma, in haplotype mapping, and as genetic markers to predict responses to drugs and adverse drug reactions. For routine clinical applications, numerous single nucleotide substitutions have been shown to be associated with disease with the HFE C282Y (haemochromatosis), apolipoprotein E4 (Alzheimer's), factor V Leiden (thrombophilia) mutations as common examples. This review summarises, in broad terms, some of the current and emerging techniques for typing of such mutations.

Restriction Enzyme Analysis

Restriction enzyme digestion of polymerase chain reaction (PCR) amplified DNA followed by electrophoresis and ethidium bromide staining was developed more than twenty years ago and remains a common genotyping method. Genotyping is based on the selectivity of restriction endonucleases for short and specific DNA sequences which are often either created or disrupted by a mutation. An example for genotyping of the HFE C282Y mutation is shown in Figure 1. Even when a mutation does not result in the creation or abolition of a restriction site, it is often possible to introduce artificial restriction sites by using mutagenic PCR primers.1

Figure 1
Genotyping for the haemochromatosis C282Y mutation by restriction enzyme analysis of PCR amplified DNA. PCR amplification results in a 390 bp product which is cleaved by the restriction enzyme Rsa I to 250 and 140 bp fragments for CC homozygotes and 250, ...

The method is very simple, requires very little in terms of instrumentation or expertise, and is generally robust. However, if many different genotyping assays are being conducted, storage of a large collection of different restriction enzymes is required, some restriction enzymes may be relatively expensive, and it is not always possible to find a suitable restriction enzyme, or to introduce an arti cial restriction site. Even if the latter is possible, it often adds constraints to the selection of PCR primers that give the best PCR product yield and specificity. Finally, the processes, such as gel electrophoresis, are not easily automated, and while some investigators have adapted this approach to higher throughput,2 these many limitations place constraints on laboratory efficiency and there is a trend for laboratories carrying out genotyping on a larger scale to use more efficient approaches, such as the homogenous assay systems described later.

Allele-Specific Amplification

Allele-specific amplification, also known as the amplification refractory mutation system (ARMS), uses allele specific oligonucleotide (ASO) PCR primers and was an early and commonly used PCR based method for genotyping.3 In this approach, one of the two oligonucleotide primers used for PCR is designed to bind to the mutation site, most commonly with the 3' end of the primer targeting the mutation site. Under carefully controlled conditions (annealing temperature, magnesium concentration etc.), amplification only takes place if the nucleotide at the 3' end of the PCR primer is complementary to the base at the mutation site, with a mismatch being “refractory” to amplification. If the 3' end of the primer is designed to be complementary to the normal gene, then PCR products should be formed when amplifying the normal gene but not genes with the mutation, and vice versa. There are numerous variations of the approach. Figure 2 shows one of the simplest embodiments where two amplifications are carried out, one using a primer specific for the normal gene, and a second using a primer specific for the mutant gene. This is followed by gel electrophoresis and ethidium bromide staining to detect the presence of amplified products. The general approach is very suitable for rapid low cost genotyping. However, when using ARMS, PCR conditions need to be very carefully controlled for accuracy. Genotyping is on the basis of the presence or absence of PCR products when using PCR primers specific for either the normal or mutant allele. However, absence of a product may also be due to sub-optimal PCR conditions or low DNA quantity or quality. Therefore it is common to include additional PCR primers for amplification of a control gene in the reaction mix, typically an unrelated gene. Absence of the PCR products of the control gene suggests sub-optimal conditions. In a variation of the approach, termed mutagenically separated PCR (MS-PCR), two ARMS primers of different lengths, one specific for the normal gene and one for the mutation are used. This yields PCR products of different lengths for the normal and mutant alleles.4 Subsequent gel electrophoresis shows at least one of the two allelic products. This approach obviates the need for two separate PCR amplifications such as described for Figure 2. Since there should always be a PCR product, either of the normal, mutant, or both (for a heterozygote) genes, this approach obviates the need to amplify a control gene.

Figure 2
Example of an ARMS genotyping assay for detection of an A to G nucleotide substitution. Results for the three possible genotypes (AA, GG and AG) are shown above the PCR products.

A variation of this forms the basis of the Masscode™ system (www.bioserve.com). The latter uses small molecular weight tags covalently attached through a photo-cleavable linker to the ARMS primer, with each ARMS primer labelled with a tag of differing molecular weight.5 A catalogue of numerous tags allows simultaneous amplification/genotyping (multiplexing) of 24 different targets in a single PCR tube. For any one mutation, genotyping is based on comparison of the relative abundance of the two relevant mass tags by mass spectrometry.

Ligation Based Assays

A number of approaches make use of DNA ligase, an enzyme that can join two adjacent oligonucleotides hybridised to a DNA template. The specificity of the approach comes from the requirement for a perfect match between the hybridised oligonucleotides and the DNA template at the ligation site. In the oligonucleotide ligation assay (OLA, Figure 3), the sequence surrounding the mutation site is first amplified, and one strand serves as a template for three ligation probes, two of these are ASOs and the third a common probe. Numerous approaches can be used for the detection of the ligated products. For example, the two ASOs can be differentially labelled with fluorescent or hapten labels and ligated products detected by fluorimetric or colorimetric enzyme-linked immunosorbent assays, respectively.6 For electrophoresis-based systems, use of mobility modifier tags or variation in probe lengths coupled with fluorescence detection enables the multiplex genotyping of several single nucleotide substitutions in a single tube.7 When used on arrays, ASOs can be spotted at specific locations or addresses on a chip. PCR amplified DNA can then be added and ligation to labelled oligonucleotides at specific addresses on the array measured.8

Figure 3
Example of an OLA for an A to G nucleotide substitution. Results for the “A” allele only are shown. PCR amplified DNA, DNA ligase and three oligonucleotide probes are added. Two of the latter are allele specific oligonucleotides (ASOs ...

Single-Base Extension

Single-base extension or minisequencing involves the annealing of an oligonucleotide primer to the single strand of a PCR product and the addition of a single dideoxynucleotide by thermostable DNA polymerase. The oligonucleotide is designed to bind one base short of the mutation site. The dideoxynucleotide incorporated is complementary to the base at the mutation site. Dideoxynucleotides do not have a free hydroxyl group at their 3' ends and once incorporated, no additional bases can be added, hence the term single-base extension. The accuracy of the approach is due to the specificity of dideoxynucleotide incorporation by DNA polymerase. This is a versatile approach and numerous chemistries and platforms can be used to detect which of the four dideoxynucleotides has been incorporated. Approaches include use of different fluorescent tags or haptens as labels for each of the four different dideoxynucleotides.9 The dideoxynucleotides differ in molecular weight and this is the basis for single-base extension methods utilising mass spectrometry; genotyping is based on the mass of the extended oligonucleotide primer, usually using matrix-assisted laser desorption/ionisation time-of fight mass spectrometry or MALDI-TOF.10 The signal output by MALDI-TOF is quantitative and can be used to calculate relative allele abundance making the approach suitable for other applications such as gene dosage studies (e.g. for estimation of allele frequencies on pooled DNA as commonly done in research applications).]

Minisequencing by MALDI-TOF, a popular choice for high throughput SNP typing by large research and industrial organisations has a low reagent cost, and is amenable to multiplexing. The approach is very cost effective for very large sample numbers and forms the basis of Sequenom’s Mass Array technology (www.sequenom.com). However, the instrumentation is very expensive and requires more specialised training than many of the other approaches described in this review. Furthermore there are a considerable number of steps following amplification, although recent developments should help to improve the efficiency of some of these post-PCR processes.11,12

ASO Hybridisation Probes

Normal or mutant alleles can also be genotyped by measuring the binding of ASO hybridisation probes. Two ASO probes, one complementary to the normal allele and the other to the mutant allele, are hybridised to PCR-amplified DNA spanning the mutation site. In early configurations, amplified products were immobilised onto a solid surface and hybridisation to radiolabelled oligonucleotides measured for a “dot blot” assay. Alternatively, for a reverse-hybridisation assay, or “reverse dot blot”, the binding of PCR products containing a quantifiable label (e.g. biotin or fluorescent labels) to solid phase allele-specific oligonucleotides can be measured. The use of microarrays comprised of hundreds of ASOs immobilised onto solid surfaces to form an array of ASOs is a common approach for large-scale genotyping of single nucleotide polymorphisms, particularly for research applications (Figure 4). Examples include the Affymetrix GeneChip® Mapping 10K Array. A fundamental requirement for accurate genotyping in such hybridisation assays is for binding of the two oligonucleotides to be highly specific for their respective alleles (mutant or normal). For this to occur, hybridisation and washing conditions need to be strictly controlled, and yet the exact conditions can vary for different mutations. This complicates matters if numerous different mutations are being typed concurrently. On high density arrays, that is microarrays used to genotype thousands of different mutation sites simultaneously, this is overcome by employing a large panel of ASOs for each mutation site. Assignment of genotypes is based on the results of all ASO probes for any particular mutation site and is usually done using a sophisticated algorithm.

Figure 4
Schematic of genotyping by hybridisation to ASOs on a micro-array slide. The example shown is for hybridisation of biotinylated PCR products to allele specific oligonucleotides for a G to A substitution. The bottom half shows results for GG and AA homozygotes, ...

Homogeneous Assays

In homogenous or “closed tube” assays, genomic DNA and all of the reagents required for amplification and genotyping are added simultaneously. Genotyping can be achieved without the need for any post-amplification processing. Commonly used examples include the 5' fluorogenic nuclease assay, also commonly known as the TaqMan® assay, and melting curve analysis of FRET probes. The methods utilise fluorogenic ASO hybridisation probes and are generally carried out on “real-time” thermocyclers, comprised of a thermocycler with an integrated fluorimeter. Changes in the fluorescence characteristics of the probes upon binding to PCR products of target genes during amplification enables “real-time” monitoring of PCR amplification, and differences in affinity of the fluorogenic probes for PCR products of normal and mutant genes enables differentiation of genotypes.

An example of the 5' fluorogenic nuclease or TaqMan® assay13 is shown in Figure 5. It has been used for genotyping of several clinically important mutations including, among others, haemochromatosis C282Y/H63D, alpha-1-antitrypsin, apolipoprotein B-100, apolipoprotein E, factor V Leiden, and prothrombin gene mutations.1416 The approach uses two dual-labelled ASO hybridisation probes complementary to the mutant and normal alleles. The two probes have different fluorescent reporter dyes, but a common quencher dye. When intact, the probes do not fluoresce due to proximity of the reporter and quencher dyes. During the annealing phase of PCR, the two probes compete for hybridisation to their target sequences, downstream of one of the primer sites, and are subsequently cleaved by the 5' nuclease activity of Thermophilus aquaticus (Taq) polymerase as the primer is extended. This results in separation of the reporter dyes from the quencher. Genotyping is determined by measurement of the fluorescence intensity of the two reporter dyes after PCR amplification (Figure 5).

Figure 5
The fluorogenic 5' nuclease or TaqMan® assay. Panel A is schematic of the principle of the assay showing the generation of fluorescence during one PCR cycle. The fluorogenic probes, comprised of ASOs with a fluorescent dye and a quencher dye, ...

Melting-curve analysis of FRET hybridisation probes is another commonly used approach, and there are numerous applications in the literature for clinically important mutations.1721 In its simplest configuration, the reaction mixture includes two oligonucleotide probes which when in close proximity form a fluorescent complex. One probe, often termed the “mutant sensor” probe, is designed to hybridise across the mutation site, and the other is an anchor probe that hybridises to an adjacent site (Figure 6A). The adjacent 3' and 5' ends of the two probes are labelled with fluorescent dyes. Fluorescent light emitted by the “donor” excites the “acceptor” fluorophore creating a unique fluorogenic complex. The complex only forms when the probes bind to adjacent sites on amplified DNA. The “sensor” probe is complementary to either the normal or mutant alleles. In the example shown, it is complementary to the C282Y allele and therefore mismatched to the normal allele. Once PCR amplification is complete, heating of the sample through the melting temperature of the probe yields a fluorescence temperature curve which differs for the normal and mutant allele (Figure 6B). This is because single-nucleotide mismatch between the sensor probe and the “normal” allele causes the probe to dissociate or melt at a lower temperature than the complex formed with the mutant allele.

Figure 6
Genotyping for the haemochromatosis C282Y mutation using FRET probes and melting-curve analysis. Panel A shows the basis of allele discrimination by melting curve analysis. The difference in stability of C and Y fluorogenic complexes results in different ...

A recent innovation obviates the requirement for fluorescent labelled probes altogether, and instead utilises a new highly sensitive fluorogenic double-stranded DNA (dsDNA) binding dye known as LCGreen™ to detect the dissociation of unlabelled ASO probes.22,23 The unlabelled ASO probes are typically 20–24 bp long and are designed to be perfectly complementary to either the mutant or normal allele. As for the FRET example above, melting curve analysis is carried out after the completion of PCR. A mismatch in the 20–24 bp ASO/template dsDNA complex results in a lower melting temperature and an earlier reduction in the fluorescent signal from the dsDNA binding dye with increases in temperature. Apart from the use of LCGreen™, the technique requires a capacity for high-resolution melting curve analysis; that is, it is not possible to carry out such analyses on all real-time thermocyclers or dedicated instruments used for melting curve analysis; clear distinction of genotypes requires exquisite temperature control, low temperature ramp rates, and high-resolution analog/digital converters to detect the very small changes in fluorescence.23,24 However, since the approach dispenses with the need for fluorescent labelled probes, it promises to simplify and reduce the cost of establishing new homogenous genotyping assays based on melting curve analysis.

The OLA previously mentioned under Ligation Based Assays, can also be performed in a homogenous system by use of FRET probes.25 The PCR/ligation mix contains PCR primers, a thermostable DNA polymerase without 5' nuclease activity (to prevent cleavage of ligation probes during the ligation phase), a thermostable DNA ligase, as well as the oligonucleotides for the ligation reaction. The ligation ASOs each have a different acceptor fluorophore and the third ligation oligonucleotide, which binds adjacently to the ASOs, has a donor fluorophore. The three ligation oligonucleotides are designed to have a lower melting temperature than the annealing temperature for the PCR primers to prevent their interference in PCR amplification. Following PCR, the temperature is lowered to allow ligation to proceed. Ligation results in FRET between donor and acceptor dyes, and alleles can be discerned by comparing the fluorescence emission of the two dyes.

There are several alternative homogeneous PCR- and hybridisation-based techniques, including, among others, molecular beacons,26 and Scorpion® probes.27 Molecular beacons are comprised of oligonucleotides that have a fluorescent reporter and quencher dyes at their 5' and 3' ends (Figure 7). The central portion of the oligonucleotide hybridises across the target sequence, but the 5' and 3' flanking regions are complementary to each other. When not hybridised to their target sequence, the 5' and 3' flanking regions hybridise to form a stem-loop structure, and there is little fluorescence because of the proximity of the reporter and quencher dyes. However, upon hybridisation to their target sequence, the dyes are separated and there is a large increase in fluorescence. Mismatched probe-target hybrids dissociate at substantially lower temperature than exactly complementary hybrids. There are a number of variations of the “beacon” approach. Scorpion® probes are similar but incorporate a PCR primer sequence as part of the probe.27 A more recent “duplex” format gives better fluorescence signal.28

Figure 7
Example of a molecular beacon probe. Hybridisation to target sequences results in an increase in fluorescence due to separation of fluorophore from quencher. The beacon probes are included in the PCR reaction mix. Fluorescence intensity, measured in the ...

Of the many methods discussed in this review, homogenous methods offer some distinct advantages, and these are steadily replacing more traditional approaches such as restriction enzyme analysis. The Table shows the techniques used for genotyping of the haemochromatosis C282Y and H63D mutations by diagnostic laboratories participating in the RCPA QAP’s Molecular Haematology program. It shows that over half (16/28) of the laboratories are using homogenous FRET/TaqMan® chemistries. Results from a european external quality control scheme are reported to show a similar trend with 45% of samples analysed by homogenous assays.24 Such approaches offer distinct advantages: there is no requirement for post-PCR processing or a requirement for post-PCR wet areas; the potential for contamination of reagents with PCR products is greatly reduced; the turnaround time can be significantly reduced; and they enable a more streamlined workflow.

Table
Methods used by participants in the RCPA QAP’s Molecular Haematology program for HFE C282Y/H63D genotyping.

We recently described an innovation which simplifies genotyping by homogenous or real-time analysis even further.20 The approach enables the analysis of whole blood, rather than extracted DNA, by real-time PCR. The method requires treatment of whole blood for several minutes with formamide prior to analysis, and was shown to be compatible with both FRET and TaqMan® chemistries as carried out on a Roche LightCycler® and an ABI Prism PE700™ respectively. This allows for very rapid analysis indeed, particularly when coupled with fast real-time thermocyclers such as the LightCycler®. Furthermore, by obviating the need for DNA extraction, the approach minimises the potential for pre-analytical errors (e.g. mislabelling of samples).

However, while they have many advantages, homogenous assays also have some disadvantages. Real-time thermocyclers are far more expensive than conventional thermocyclers. For any one mutation, access to numerous samples of all genotype categories is often required for assay optimisation. It can also be difficult to optimise probe design and PCR conditions such that distinction of genotypes is unequivocal. Fluorescent probes are expensive resulting in high set up costs in development of new assays particularly if redesign of poorly performing probes is required. Nevertheless, there are numerous methods for clinically relevant mutations described in the literature and once assays are established they are generally reliable. In addition, and as previously mentioned, development of dyes such as LCGreen™ and high resolution melting curve analysis which obviate the need for fluorescent labelled probes promise to both simplify and reduce the cost of developing new assays for at least one category of homogenous genotyping assays.22,23

Fluorescence Polarisation (FP)

In FP, the degree to which the emitted light remains polarised in a particular plane is proportional to the speed at which the molecules rotate and tumble in solution. Under constant pressure, temperature, and viscosity, FP is directly related to the molecular weight of a fluorescent species. Hence, when a small fluorescent molecule is incorporated into a larger molecule, there is an increase in FP. Most clinical chemists are familiar with the use of FP, for example, in assays used for therapeutic drug monitoring. The general principle can be applied to any small fluorescently labelled molecule, such as a deoxynucleotide, which either dissociates or associates to a large molecular weight species. The technique can be applied to many of the chemistries already described.29,30 The 5' nuclease assay described previously, where an oligonucleotide probe is digested to a lower molecular weight species, for example, is amenable to analysis by FP, but with the added benefit of not requiring a quencher. Perkin-Elmer’s AcycloPrime -FP SNP Detection Kit is an example of a FP minisequencing method. Following PCR amplification, unincorporated primers and nucleotides are degraded enzymatically, the enzymes heat inactivated, and a minisequencing reaction using DNA polymerase and fluorescent-labelled dideoxynucleotides performed. FP is then measured, typically using black 96- or 384-well plates on an FP plate reader.

Pyrosequencing™

PyrosequencingTM is a novel and rapid sequencing technique. It is a homogenous method which is not based on chain termination, does not use dideoxynucleotides, and nor does it require any electrophoresis.3133 The approach is based on the generation of pyrophosphate whenever a deoxynucleotide is incorporated during polymerisation of DNA, for example, as nucleotides are added to the 3' end of a sequencing primer, or primer extension: DNAn + dNTP → DNAn+1 + pyrophosphate. The generation of pyrophosphate is coupled to a luciferase catalysed reaction resulting in light emission (Figure 8) if the particular deoxynucleotide added is incorporated, yielding a quantitative and distinctive pyrogram.

Figure 8
Indicator reaction for the Pyrosequencing™ reaction (A), and example of a Pyrosequencing™ assay for a G to A substitution on a PCR amplicon (B). For panel A: APS, Adenosine 5 phosphosulfate. PPi, pyrophosphate. For panel B, the complementary ...

Sample processing includes PCR amplification with a biotinylated primer, isolation of the biotinylated single strand amplicon on streptavidin coated beads (or other solid phase) and annealing of a sequencing primer (Figure 8). Samples are then analysed by a PyrosequencerTM [www.pyrosequencing.com] which adds a number of enzymes and substrates required for the indicator reaction, including sulfurylase and luciferase, as well as apyrase for degradation of unincorporated nucleotides. The sample is then interrogated by addition of the four deoxynucleotides. Light emission is detected by a charge coupled device camera (CCD) and is proportional to the number of nucleotides incorporated. Results are automatically assigned by pattern recognition.

Advantages of the approach include: a) the ease of assay design b) the relatively low cost of developing new assays c) the high information content of the results produced (quantitative sequencing signals) and d) automatic genotyping assignment. The approach requires less expertise than traditional sequencing methods or minisequencing using approaches such as MALDI-TOF. The quantitative sequence data generated greatly facilitates assay validation, and also makes the approach highly suitable for gene-dosage studies, including estimation of gene frequencies on pooled DNA. While assay design is relatively straightforward, protocols for numerous clinically relevant genotyping assays have been published.3438 A number of assays are also available in kit form. The instrumentation is available in two basic configurations, one suitable for sequencing, typically up to 200 bp, and the other, which utilises considerably lower reagent volumes (~1/3), is typically used to sequence 8–10 bp segments. Overall Pyrosequencing™ is a highly flexible approach suitable for a number of genetic analyses including genotyping, sequencing, as well as quantitative genetic analyses such as estimation of gene dosage. Its major disadvantage is the cost of the reagents and a very limited capacity for multiplexing.

Choosing a Genotyping Technology

This review has discussed a number of different methods for typing of clinically relevant single nucleotide substitutions. There are however, many other techniques which may also be suitable, including those not based on PCR, such as the Invader® assay.3941 The question of which genotyping technology to use is relatively complex and requires numerous considerations including the number and variety of genotyping tests, amenability of the approach to automation, for example through the use of liquid handling systems, reliability of the chemistry/instrument, cost of instrumentation and reagents, maintenance costs, technical support from manufacturers (particularly for complex instruments), throughput, turn-around time, and the availability of well trained and experienced personnel. The latter is particularly important. Almost all of the assays described can be made to work well; all can give poor results in the wrong hands. Many of the methods described are in use in large scale research and industrial projects. In practice however, the number used for diagnostic purposes are relatively few. Of the commonly used techniques, gel-based genotyping assays such as ARMS and restriction enzyme analysis are labour intensive but are relatively straightforward. They are commonly used as an entry-point in establishing molecular diagnostic techniques, are simple to develop and are useful when dealing with a small number of samples. Although such methods remain in use in many laboratories, they are cumbersome if sample numbers are high or if there are a wide variety of different genotyping assays to perform. Technologies such as Pyrosequencing and minisequencing by MALDI-TOF are highly flexible platforms with particularly roles in specialist laboratories and core genetic facilities. However for most routine purposes, homogenous chemistries offer many advantages and are available in numerous configurations. They dispense with the need for any post-PCR processing; analysis time is greatly reduced; the risk of contamination of work areas with PCR products is minimised; and they are amenable to high throughput. Finally, and as previously discussed, some homogenous approaches also have the potential to be adapted for analysis of whole blood thus dispensing with the need for DNA extraction.

Future Trends and Conclusion

There is a vast array of chemistries and platforms which can be used for typing of single nucleotide substitutions. However no one technology is clearly superior and it is not unusual for laboratories to use various approaches for different genotyping tests. However, on the basis of their many advantages, it seems highly likely that homogenous genotyping technologies will continue to be developed, and in one guise or another, these are likely to remain a dominant category. As the number of genetic loci found to be associated with disease (and possibly drug response) continues to expand, the ability to conduct a large number of genotyping assays in a single tube, or multiplexing, will become increasingly important. On that basis, non-homogenous technologies which are much more amenable to very high orders of multiplexing, such as micro-arrays and minisequencing by MALDI-TOF, are also expected to play an important role.

Acknowledgments

I am grateful to John Ivey and Melinda Higgins, Department of Haematology, Division of Laboratory Medicine, Pathwest, Royal Perth Hospital, Perth, WA, for data used in Figure 5, on factor V Leiden genotyping.

Footnotes

Competing interests: None declared

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