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1 Department of Applied Genomics, Genometrix Inc., The Woodlands, TX 77381, and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109.
Abstract
Background: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients’ genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge.
Approach: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs).
Content: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan®, and molecular beacon genotyping. Rolling circle amplification and InvaderTM assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena.
Summary: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.
Recent research demonstrates that certain genetic polymorphisms cause significantly different responses among individuals on exposure to a particular drug (1)(2). Pharmacokinetic variations in absorption, distribution, metabolism, and excretion of therapeutic agents have been studied extensively during the past two decades (2). More recently, pharmacodynamic variations, including receptor and transporter polymorphisms, also have also been shown to cause individual variations in drug responses (1). Understanding the role of genetic polymorphisms in drug responses will help to increase drug efficacy and decrease adverse effects by tailoring medications to patients’ genetic make-up. Advances in this area have important clinical implications and practical value for the design of dosing regimens. When applied to drug discovery and development, pharmacogenetics may help the development of therapeutic interventions targeting specific responder patient groups (3).
Several types of variants exist in the human genome, including single nucleotide polymorphisms (SNPs),1 microsatellite, or insertion/deletion polymorphisms. SNPs are single-base variations at a unique physical location. Microsatellites are tandem repeats of multiple copies of the same base sequence motif on a chromosome. These polymorphisms are used extensively as markers in linkage analysis, i.e., studies of the tendency of certain genes to be inherited together in families.
Although microsatellites have been used in linkage analysis in the past, it is becoming increasingly popular to use SNP markers because they are the most frequent DNA sequence variations found in the human genome. Genotyping large number of SNPs in linkage and association studies will shed new light to the understanding of complex disease traits, including many common human diseases and drug responses (3). SNP genotyping is also easier to automate because SNPs can be screened in a digital format by analyzing the presence or absence of a sequence. The SNP Consortium was created in 1999 by pharmaceutical companies and the Wellcome Trust to develop a catalog of 300 000 SNPs for public usage. It is projected that the consortium will generate 1 000 000 SNPs by the end of the first quarter of year 2001 (Arthur Holden, personal communication). The high-density SNP map will allow researchers to expand their capabilities for identification of critical diseases and drug response genes in nonfamilial studies.
With the completion of the human genome map and the SNP map, genotyping SNPs in large-scale pharmacogenetic studies will be an integrated part of the drug discovery and development process. Advanced technologies to identify genetic polymorphisms rapidly, accurately, and economically are becoming a priority in the implementation of pharmacogenetics to drug development, clinical trials, and clinical monitoring for drug efficacy and toxicity. Pharmacogenetics will have a major impact on the healthcare and medical practice by enabling selection of the right drugs for the right patients at the right doses through genetics-based diagnostic tests (3). In this article, I review the current technological progress on mutation detection and genotyping with an emphasis on high-throughput technologies for SNP genotyping.
Identification of New Polymorphisms
A variety of techniques are available for new mutation/polymorphism identification. One commonly used strategy for SNP screening is to amplify genes of interest by PCR, scan the PCR products for the presence of DNA variants by confirmation-based mutation scanning methods, and then sequence positive PCR products. The development of new-generation DNA sequencers also allows direct heterozygote sequencing frequently used in SNP identification. With a vast amount of human expressed sequence tags (ESTs) and genomic clones in the public domain, computer-based sequence alignment and clustering also provide a rich source for SNP identification.
conformation-based mutation scanning
Single-strand conformation polymorphism analysis is one of
the most widely used methods for mutation detection. In single-strand
conformation polymorphism, DNA regions with potential polymorphisms are
first amplified by PCR. Single-stranded DNAs are then generated by
denaturation of the PCR products and separated on a nondenaturing
polyacrylamide gel. A fragment with a single base modification
generally forms a different conformer and migrates differently when
compared with wild-type DNA (Fig. 1
) (4)(5). The sensitivity can be
increased to nearly 100% by coupling with either restriction enzyme
fingerprinting (6) or dideoxy-sequencing fingerprinting
(7).
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Other alternative conformation-based mutation screening methods include conformation-sensitive gel electrophoresis (8), chemical or enzymatic mismatch cleavage detection (9)(10)(11), denaturing gradient gel electrophoresis (12), and denaturing HPLC (13). The underlying principle of these methods is that the melting characteristics of double-stranded DNA are largely defined by its sequence. Therefore, a single-base mismatch can produce conformation changes in the double helix that cause the differential migration of homoduplexes and heteroduplexes containing base mismatches during gel electrophoresis. Conformation-sensitive gel electrophoresis has been developed to distinguish homoduplexes from heteroduplexes containing a single mismatched base pair by polyacrylamide gel electrophoresis in a mildly denaturing solvent system. This method has been shown to be highly sensitive for identifying mutations in areas of highly repetitive and GC-rich sequences (14).
Mismatch cleavage detection takes advantage of the fact that mismatched bases are sensitive to cleavage by enzymes or chemicals. After PCR amplification, the wild-type and variant products are subjected to denaturation/renaturation to create heteroduplex molecules. After incubation with resolvases or chemicals, the products are resolved electrophoretically side by side to score for the presence of mismatch-cleaved molecules (9)(10)(11).
In denaturing gradient gel electrophoresis, genomic DNA is PCR amplified and the products are resolved in a polyacrylamide gel with an increasing denaturing gradient of formamide and urea under careful temperature control. Heteroduplex DNA fragments with a single mismatched base pair are revealed by migrational differences from homoduplexes. The advantages of this method are its high accuracy and relatively low costs. The disadvantages are low throughput and difficulty of optimization (12).
Denaturing HPLC detects polymorphisms by analyzing the DNA mobility of different heteroduplexes using chromatography in a slightly denaturing condition. The WAVE® DNA analysis system is a commercialized method from Transgenomic, Inc. (San Jose, CA) that uses temperature-modulated heteroduplex analysis (15). The variant sample first is hybridized with wild-type DNA to form a mixture of homo- and heteroduplexes. The heteroduplexes can be separated from the homoduplexes by column chromatography at a temperature that partially denatures the mismatched DNA. The advantage of using the WAVE system is that multiple samples could be pooled into a single reaction for variant detection. Therefore, it can substantially increase the throughput compared with DNA sequencing.
direct dna sequencing
Conformation-based mutation screening tools are a cost-effective
way to detect novel polymorphisms. Once the potential regions have been
confirmed to contain putative polymorphisms, these regions can be
sequenced to locate the final polymorphic sites (Fig. 2
). With the improvement of computer software and detection
systems, fluorescent DNA sequencing has become fully automated for the
DNA sequencing process. The SNP Consortium has been identifying SNPs
using this technology by sequencing and comparing multiple
individuals’ sequences. The 3700 genetic analyzer (Applied Biosystems,
Foster City, CA) and MegaBACETM (Amersham
Pharmacia Biotech, Uppsala, Sweden) systems are fully automated
sequencers that are emerging as vital technology platforms for
large-scale DNA sequencing projects.
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in silico snp mapping
The Human Genome Project has produced large amounts of sequence
data in the public databases. These EST and genomic DNA sequences
provide a rich source for in silico identification of SNPs. Through
fragment clustering and multiple alignment of the sequences from
redundant EST and bacterial artificial chromosome clones, potential
SNPs can be identified without the initial effort to sequence multiple
individuals. A computer SNP screening algorithm called POLBAYES was
developed and is available at no cost for nonprofit usage
(16) (instructions at
http://genome.wustl.edu/gsc/polybayes). By calculating the
probability scores, putative SNPs can be identified. These putative
SNPs can be confirmed by resequencing in multiple individuals.
Obviously, this is a fast and cost-efficient way to identify new SNPs.
Gel-based Genotyping Methods for Detecting Known Polymorphisms
pcr-restriction fragment length polymorphism analysis
Commonly used methods include gel electrophoresis-based techniques
such as PCR coupled with restriction fragment length
polymorphism analysis. Specific regions of DNA sequences can be
PCR amplified. The PCR products then are digested with appropriate
restriction enzymes and visualized by staining the gel after
electrophoresis. If the genetic polymorphism produces a gain or loss of
the restriction site, a different restriction digestion pattern can be
recognized (17). A major limitation of the
PCR-restriction fragment length polymorphism method is the requirement
that the polymorphisms alter a restriction enzyme cutting site
(17).
oligonucleotide ligation assay genotyping
The oligonucleotide ligation assay (OLA) relies on hybridization
with specific oligonucleotide probes that can effectively discriminate
between the wild-type and variant sequences. Three oligonucleotides are
used in OLA: two allele-specific oligonucleotide probes (one specific
for the wild-type allele and the other specific for the mutant allele)
plus a fluorescent common probe. The 3' ends of the allele-specific
probes are immediately adjacent to the 5' end of the common probe. The
gene fragment containing the polymorphic site is amplified by PCR and
incubated with the probes. In the presence of thermally stable DNA
ligase, ligation of the fluorescent-labeled probe to the
allele-specific probe(s) occurs only when there is a perfect match
between the variant or the wild-type probe and the PCR product
template. These ligation products are then separated by
electrophoresis, which permits the recognition of the wild-type
genotypes, the variants, the heterozygotes, and the unligated probes
(Fig. 3
). By varying the combinations of color dyes and probe lengths,
multiple mutations can be detected in a single reaction
(18). This method can be used to genotype a large panel of
informative biallelic markers. The hybridization of allele-specific
oligonucleotides is dependent on both the variant and the surrounding
sequences. Highly GC-rich DNA regions make the allele-specific ligation
step in OLA difficult to optimize and multiplex.
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minisequencing
Similar to regular DNA sequencing, minisequencing is an efficient
way to detect SNPs through the addition of specific nucleotides to a
single primer (19). Several SNP markers can be analyzed in
parallel by the use of locus-specific primers and analyzing the
allele-specific incorporation of labeled nucleotides (20).
To increase the throughput, new sets of reaction products can be loaded
onto the same gel.
Overall, gel-based genotyping assays are relatively straightforward and are useful when dealing with a small number of samples. The methods are labor-intensive and require experienced and skilled technical staff for final analysis. Although gel-based genotyping methods are still widely used in many laboratories, they are difficult to apply to high-throughput genotyping in large-scale pharmacogenetic studies.
Non-Gel-based High-Throughput Genotyping Technologies
Non-gel-based high-throughput genotyping technologies are rapidly evolving as the dominant genotyping platforms for large-scale pharmacogenetic studies. Current genotyping methods include fluorescent dye-based high-throughput genotyping procedures such as homogeneous solution hybridization, allele-specific OLA and minisequencing. High-density chip array and mass spectrometry (MS) similarly have been used for genotyping both known and unknown polymorphisms.
homogeneous solution hybridization using fluorescence resonance
energy transfer detection
Fluorescence resonance energy transfer (FRET) occurs when
two fluorescent dyes are in close proximity to one another and the
emission spectrum of one fluorophore overlaps the excitation spectrum
of the other fluorophore (21). Commonly used FRET-based
technologies include the TaqMan® assay and
molecular beacons. The InvaderTM assay is a new
technology enabling genotyping SNPs without PCR.
TaqMan genotyping.
The TaqMan Allelic Discrimination assay
uses the 5' nuclease activity of Taq polymerase to detect a
fluorescent reporter signal generated during or after PCR reactions
(22). For SNP genotyping, one pair of TaqMan probes and one
pair of PCR primers are used. The assay uses two TaqMan probes that
differ at the polymorphic site, with one probe complementary to the
wild-type allele and the other to the variant allele. A 5' reporter dye
and a 3' quencher dye are covalently linked to the wild-type or variant
allele probes. When the probes are intact, fluorescence is quenched
because of the physical proximity of the reporter and quencher dyes.
During the PCR annealing step, the TaqMan probes hybridize to the
targeted polymorphic site. During the PCR extension phase, the 5'
reporter dye is cleaved by the 5' nuclease activity of the
Taq polymerase, leading to an increase in the characteristic
fluorescence of the reporter dye. Specific genotyping is determined by
measuring the signal intensity of the two different reporter dyes after
the PCR reaction (Fig. 4
). In addition to detecting SNPs (23), small gene
deletions and insertions can also be identified by this method
(24). TaqMan genotyping instrumentation and reagents are
supported by Applied Biosystems.
|
Molecular beacons.
Molecular beacons are oligonucleotide
probes that have two complementary DNA sequences flanking the target
DNA sequence and a donor-acceptor dye pair at opposite ends of each
probe. When not hybridized to the target, the probe adopts a
hairpin-loop conformation with the reporter and quencher dyes close
together, and therefore, no donor fluorescence is generated. When
hybridized to the right target sequence, the two dyes are separated and
the fluorescence is increased dramatically (25). Mismatched
probe-target hybrids dissociate at substantially lower temperature than
exactly complementary hybrids. This thermal instability of mismatched
hybrids increases the specificity of molecular beacons. For SNP
genotyping, two molecular beacons with exact sequence matches to the
wild-type and variant alleles are used in the same PCR reaction. These
two molecular beacons are labeled with different fluorophores that emit
fluorescent light at distinct optical wavelengths. The use of two
differentially labeled molecular beacons in the same PCR reaction
allows simultaneous determination of three possible allelic
combinations. The molecular beacon technology has been commercialized
by Stratagene (La Jolla, CA).
Invader assay.
The Invader assay (Third Wave Technologies,
Inc., Madison, WI) is an attractive FRET-based genotyping method with
the potential to genotype SNPs without PCR amplification. Two
oligonucleotides, a wild-type or variant signal probe plus an upstream
Invader probe, are used in each reaction. These probes hybridize in
tandem to a specific region of genomic DNA. When the 3' end of an
upstream oligonucleotide overlaps the hybridization site of the 5' end
of a downstream oligonucleotide probe by at least one base pair, the
structure will be recognized and cleaved by
Cleavase® enzymes, a class of naturally
occurring and engineered enzymes (26)(27). A
single-nucleotide mismatch immediately upstream of the cleavage site
renders the confirmation unrecognizable by Cleavase. Invader assays are
conducted isothermally, and a linear increase in signal over time will
be produced (Fig. 5
). Each cleavage product then serves as an Invader
oligonucleotide in a secondary reaction, where it directs the cleavage
of a combined labeled FRET probe-template construct. This secondary
oligonucleotide probe is 5' end-labeled with the donor fluorophore,
which is quenched by an internal acceptor dye. When the DNA is cleaved,
the donor and acceptor dyes are no longer in close proximity, the
quenching is abolished, and fluorescence is generated. Assays are read
with a fluorescence plate reader, and genotypes are assigned after
determination of the net wild-type/variant signal ratio for each sample
(26)(28).
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FRET-based genotyping approaches are fast and often can be monitored in real time. In addition, these assays can use standard microtiter plates in 96- to 1536-well formats. The ability to automate data handling further enhances accuracy by eliminating operator bias. These assays are robust for large-scale characterization of the same genetic polymorphism. Both TaqMan and molecular beacon assays require dual-labeled probes and PCR reactions, which are likely more expensive than the Invader technology.
Allele-specific ligation
Dye-labeled oligonucleotide ligation.
Dye-labeled
oligonucleotide ligation combines PCR-OLA with FRET detection in a
one-step homogeneous assay. The PCR primers used in the assay are
designed to have high melting temperatures. The three dye-labeled
ligation probes for each SNP are designed to have low melting
temperatures. A 5' donor dye-labeled common probe terminates one base
immediately upstream from the polymorphic site. Two
allele-specific 5'-phosphorylated, 3'-acceptor dye-labeled probes
have polymorphic nucleotides at the 5' end. A thermostable DNA
polymerase with no 5' nuclease activity (AmpliTaq FS) and a
thermostable DNA ligase are used. The first stage of PCR reaction is
kept at high temperature, and the ligation probes are unable to anneal.
After sufficient PCR products are generated, the second stage of the
reaction, with a low annealing temperature, allows ligation to occur.
By analyzing the fluorescence signals of all the dyes, individual
genotypes can be determined directly after one reaction using real-time
PCR or by end-point signal analysis using a fluorescent plate reader
(29).
Rolling circle amplification (RCA).
Like the Invader assay,
RCA is also sensitive enough to work directly from genomic DNA
(30)(31). For each SNP, two allele-specific
probes are designed to discriminate between the two alleles. Each probe
consists of a single oligonucleotide 80–90 bases in length. The 5' end
of the probe is phosphorylated and bears a sequence of 20 nucleotides
that is complementary and, therefore, will hybridize to the region
immediately 5' of the SNP. The 3' end of the probe contains 10–20
nucleotides complementary to the region immediately 3' of the SNP. Both
allele-specific probes are identical with the exception of the 3' base,
which is varied to complement the two alleles at the polymorphic site.
The two probes contain two different generic backbone sequences. A
generic backbone that encodes binding sites for two RCA amplification
primers is sandwiched between the allele-specific probe arms. The first
stage of the assay involves ligation of the probes to the target DNA by
allelic discrimination. After denaturation of the target genomic DNA,
both allele-specific probes are then added to the denatured DNA. A
stable hybrid is formed only if the 3' base of the probe is perfectly
matched to the polymorphic nucleotide present in the target. Complete
hybridization aligns the two ends of the probe together on the target
DNA and circularizes the probe by a thermostable ligase. Amplification
of a circularized probe by RCA requires the first primer to hybridize
to its complementary region on the probe backbone. In the presence of a
strand-displacing DNA polymerase, the primer is extended, eventually
displacing itself at its 5' end once one complete revolution of the
circularized probe is made. Continued polymerization and displacement
generates a single-stranded, concatameric DNA copy of the original
probe. The isothermal process is amenable to automation, and the
generic amplification primers keep reagent costs low. A FRET-based RCA
assay called SNIPERTM has been developed and is
in the initial stage of commercialization by Amersham Pharmacia
Biotech.
allele-specific nucleotide incorporation
Pyrosequencing.
Pyrosequencing detects de novo incorporation
of nucleotides based on the specific template. The incorporation
process releases a pyrophosphate, which is converted to ATP in the
presence of adenosine 5'-phosphosulfate, which in turn stimulates
luciferase. The light production in the luciferase-catalyzed reaction
is detected by a charge couple device camera. The height of each peak
correlates to the light signal and is proportional to the number of
nucleotides incorporated. ATP and unincorporated dNTPs are continuously
degraded by apyrase. The light is switched off, and the next dNTP is
added. As the process continues, the complementary DNA strand is built
up and the nucleotide sequence is determined from the signal peak in
the pyrogram. Current instrumentation, sold by Pyrosequencing AB
(Uppsala, Sweden), can detect 500 SNPs/h post-PCR in a 96-well format
(32).
Single-base extension with fluorescence detection.
A popular
SNP genotyping method is the template-directed dye terminator
incorporation assay with fluorescent polarization (FP) detection. FP is
based on the principle that a small molecule tumbles rapidly in
solution. If plane-polarized light is shone on fluorescent dye labels,
the molecules tumble rapidly, and the emission is depolarized. If the
viscosity and temperature are constant, FP is directly proportional to
the molecular volume, which is directly proportional to the molecular
weight (33). The sequencing primer is immediately upstream
from the polymorphic site. When incubated in the presence of
dideoxynucleotide triphosphates labeled with different
fluorophores, the allele-specific dye-labeled dideoxynucleotide
triphosphate is linked to the primer in the presence of DNA
polymerase and target DNA. The genotype of the target DNA molecule can
be determined simply by exciting the fluorescent dye in the reaction
and determining the change in FP (33). LJL Biosystem
(Sunnyvale, CA) has developed a high-throughput genotyping system using
this technology.
dna microarray genotyping
The DNA microarray is a hybridization-based genotyping technique
that offers simultaneous analysis of many polymorphisms. High-density
microarrays are created by attaching hundreds of thousands of
oligonucleotides to a solid silicon surface in an ordered array. The
DNA sample of interest is PCR amplified to incorporate fluorescently
labeled nucleotides and then hybridized to the array. Each
oligonucleotide in the high-density array acts as an allele-specific
probe. Perfectly matched sequences hybridize more efficiently to their
corresponding oligomers on the array and, therefore, give stronger
fluorescent signals over mismatched probe-target combinations. The
hybridization signals are quantified by high-resolution fluorescent
scanning and analyzed by computer software. DNA alterations such as
heterozygous base-pair polymorphisms or mutations, insertions, and
deletions can be identified (34)(35).
Sequencing by hybridization can be an efficient method to monitor a large number of SNPs. It is possible to array a set of short oligonucleotides covering the entire DNA fragment on a DNA chip. Because the precise sequence of the oligonucleotide at each location on the chip is known, the pattern of hybridization can be determined using fluorescently labeled DNA probes. The advantage of this method is that a large DNA fragment or a large collection of small PCR products can be scanned in one hybridization. More than 100 such arrays have been used for high-throughput screening of SNPs covering more than 2 Mb of genomic DNA (36) and thousands of SNPs have been screened rapidly by use of chip-based resequencing (37).
Once a high-density SNP map is generated, application of
microarray technology to simultaneously monitor tens of thousands of
genetic variations will provide major advantages for genome-wide
screening. The application of these chip-based genotyping technologies
is currently under development and evaluation in several companies,
including Affymetrix (Santa Clara, CA), Protogene (Menlo Park, CA),
Genometrix (The Woodlands, TX), and Motorola BioChip Systems
(Northbrook, IL). Several arrays have been generated to detect variants
in the HIV genome, p53, human mitochondria mutations, and human
cytochrome p450 polymorphisms. The current limitation is that
homozygous variants are readily detected but heterozygous may be
missed, and deletions and insertions will not be specifically
recognized. Although expensive now, the cost of chips as a major
screening platform in pharmacogenetics will likely be reduced with
analyses of hundreds of thousands of patients. Genometrix has developed
a low-cost, medium-density array format using a multiplex capillary
printer and high-speed robotics. Each array contains up to 256
elements, and 96 arrays can be processed in parallel on a single plate.
For SNP genotyping, wild-type and variant-specific probes are printed
side by side in each array. Target sequences containing the
polymorphisms of interest are derived by multiplex PCR reaction from
genomic DNA sequences. By analyzing the fluorescent signals after
hybridization, multiple SNPs can be genotyped in a single array (Fig. 6
) (38).
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ms
Large molecules can be identified by MS through electrospray or
matrix-assisted laser desorption ionization and ion-trap or
time-of-flight detectors. MS yields precise information on the
molecular mass of the DNA fragments. The procedure can be fully
automated, and both DNA strands can be analyzed in parallel
(39). Unlike the fluorescent genotyping methods described
above, MS offers specificity and accuracy without requiring specially
labeled probes or primers. One disadvantage of this technique is that
it requires purified samples free of ions and other impurities, thus
increasing the technical time and sample-processing costs. A chip-based
genotyping method using MS has been described (40). PCR can
be performed in 1-µL reactions directly in the chip wells in
parallel. The PCR product can be detected in situ using matrix-assisted
laser desorption ionization MS. This miniaturization technique has the
potential for high-throughput, low-cost genotyping. The MassARRAY
instrumentation for SNP genotyping has been commercialized by Sequenom,
Inc. (La Jolla, CA).
Conclusions and Perspectives
A major challenge for large-scale pharmacogenetic studies is to compare hundreds of thousands of polymorphisms among numerous individuals. Thus, the success of pharmacogenetics depends on user-friendly technologies that can detect polymorphisms rapidly, accurately, and cost effectively on a large scale. Each of the methods discussed above has been demonstrated to work in a variety of settings, but each method has its limitations. Of all the options available, FRET-based and chip-based genotyping technologies will most likely evolve as the ultra-high-throughput detection systems to meet the requirements of pharmacogenetics. For DNA microarrays, a better approach is to fabricate a generic array containing tag sequences close to the polymorphic sites. Genotyping can be conducted by use of fluorescently labeled single-base extension reactions or OLAs. For discovery work in pharmacogenetics, large numbers of SNPs can be genotyped using high-density arrays. Once the association has been established, a limited number of SNPs probably will be included in routine clinical trials or clinical practice using medium-density array genotyping. With advanced genotyping technologies, pharmacogenetics will revolutionize our concepts of modern medicine by allowing physicians to prescribe the most effective and safest drug based on a patient’s genetic blueprint.
Footnotes
Address for correspondence: Genometrix Inc., 2700 Research Forest Dr., The Woodlands, TX 77381. Fax 281-465-5001; e-mail mshi{at}genometrix.com.
1 Nonstandard abbreviations: SNP, single nucleotide polymorphism; EST, expressed sequence tag; OLA, oligonucleotide ligation assay; MS, mass spectrometry; FRET, fluorescence resonance energy transfer; RCA, rolling circle amplification; and FP, fluorescent polarization. 
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 A. Yokoyama, T. Yokoyama, T. Muramatsu, T. Omori, S. Matsushita, S. Higuchi, K. Maruyama, and H. Ishii Macrocytosis, a new predictor for esophageal squamous cell carcinoma in Japanese alcoholic men Carcinogenesis, November 1, 2003; 24(11): 1773 - 1778. [Abstract] [Full Text] [PDF]
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E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+-cyclen complex Nucleic Acids Res., November 15, 2002; 30(22): e126 - e126. [Abstract] [Full Text] [PDF]
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A. Simeonov and T. T. Nikiforov Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection Nucleic Acids Res., September 1, 2002; 30(17): e91 - e91. [Abstract] [Full Text] [PDF]
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P. S. Bernard and C. T. Wittwer Real-Time PCR Technology for Cancer Diagnostics Clin. Chem., August 1, 2002; 48(8): 1178 - 1185. [Abstract] [Full Text] [PDF]
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H. Millward, W. Samowitz, C. T. Wittwer, and P. S. Bernard Homogeneous Amplification and Mutation Scanning of the p53 Gene Using Fluorescent Melting Curves Clin. Chem., August 1, 2002; 48(8): 1321 - 1328. [Abstract] [Full Text] [PDF]
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J. Pickering, A. Bamford, V. Godbole, J. Briggs, G. Scozzafava, P. Roe, C. Wheeler, F. Ghouze, and S. Cuss Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms Nucleic Acids Res., June 15, 2002; 30(12): e60 - e60. [Abstract] [Full Text] [PDF]
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M.-Z. Xue, O. Bonny, S. Morgenthaler, M. Bochud, V. Mooser, W. G. Thilly, L. Schild, and P.-M. Leong-Morgenthaler Use of Constant Denaturant Capillary Electrophoresis of Pooled Blood Samples to Identify Single-Nucleotide Polymorphisms in the Genes (Scnn1a and Scnn1b) Encoding the {alpha} and {beta} Subunits of the Epithelial Sodium Channel Clin. Chem., May 1, 2002; 48(5): 718 - 728. [Abstract] [Full Text] [PDF]
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X. Qi, S. Bakht, K. M. Devos, M. D. Gale, and A. Osbourn L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs) Nucleic Acids Res., November 15, 2001; 29(22): e116 - e116. [Abstract] [Full Text] [PDF]
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