current methods for high-throughput detection of novel dna polymorphisms
TRANSCRIPT
TECHNOLOGIES
DRUG DISCOVERY
TODAY
Drug Discovery Today: Technologies Vol. 3, No. 2 2006
Editors-in-Chief
Kelvin Lam – Pfizer, Inc., USA
Henk Timmerman – Vrije Universiteit, The Netherlands
Genomic technologies
Current methods for high-throughputdetection of novel DNApolymorphismsThomas Peters, Reinhard Sedlmeier*Ingenium Pharmaceuticals AG, Fraunhoferstr. 13, 82152 Martinsried, Germany
For research varying from the identification of specific
disease loci to the investigation of protein function, the
detection of DNA sequence variations requires reliable
methods. Technologies enabling rapid and cost effec-
tive identification of novel genetic polymorphisms will
significantly impact future work in genetic mapping
studies, drug target discovery and validation and phar-
macogenomics.
*Corresponding author: R. Sedlmeier ([email protected])
1740-6749/$ � 2006 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2006.05.002
Section Editor:Andreas Russ – University of Oxford, Oxford, UK
Introduction
The completion of genome sequences from many species
boosted activities in the identification and analysis of genetic
variants. For such enterprises a broad spectrum of technolo-
gies is available which allow accurate and efficient detection
of sequence polymorphisms. Within this technological range
a distinction can be made between methods for the identi-
fication of known polymorphism, for example, as required for
genetic mapping and association studies (genotyping), and
methods for the detection of novel polymorphisms (muta-
tion scanning). This review is focused on mutation scanning
and emphasis is laid on techniques, which combine reliabil-
ity and throughput. The described methods are suited for
mutation screens on large genomic regions, where changing
sets of genes are analyzed for mutations, and/or for the
detection of novel alleles in large sample numbers on a
limited set of target genes.
Mutation detection assay principles
For the detection of novel point mutations or small deletions
and insertions several physical or enzymatic principles can be
used to reflect the variation in DNA sequence:
� M
ethods based on variations in electrophoretic mobilityinduced by conformational differences in single or double-
stranded DNA molecules.
� M
ethods based on DNA conformation induced retentiontime differences in ion-pair reverse-phase chromatography
– denaturing high performance liquid chromatography
(DHPLC).
� M
ethods based on recognition and cleavage of mismatchesin HETERODUPLEX (see Glossary) molecules by specific endo-
nucleases and separation of the cleavage products.
� M
ethods based on recognition and repair of mismatches ina biological system for selection and amplification of the
mutant allele sequence – mismatch repair detection (MRD).
� M
ethods based on nucleotide-specific enzymatic orchemical fragmentation followed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS).
Conformation based electrophoretic separation
methods
The basis for detecting mutations in double-stranded (ds)
DNA during electrophoresis is the fact that the melting
123
Drug Discovery Today: Technologies | Genomic technologies Vol. 3, No. 2 2006
Glossary
Heteroduplex: mismatch containing DNA fragment (plural:
heteroduplices) generated from a mixture of wild-type and mutant
fragments by heat denaturing and re-annealing.
Homoduplex: perfectly base-paired DNA fragment (plural:
homoduplices).
Figure 1. Schematic overview of the described methods for high-throughput
mismatch detection in heteroduplex DNA. Heteroduplices are separated from
(example shown for TGCE) or differences in retention behavior (DHPLC). Alter
and fluorophore-labeled cleavage products are size separated. (B) Mismatch R
biological system (E. coli), thus enabling the selection and amplification of mutant a
specific fragmentation of target sequences followed by pattern analysis of the frag
electrophoresis; DGGE: denaturing gradient gel electrophoresis; DHPLC: denat
assisted laser desorption/ionization time-of-flight mass spectrometry.
124 www.drugdiscoverytoday.com
temperature (Tm) of dsDNA is completely sequence depen-
dent. A 1 bp sequence difference in two HOMODUPLEX molecules
(see Glossary) can change the Tm of the respective DNA
domain by up to 1 8C or more and a mismatch in hetero-
duplices alters Tm by up to 6 8C compared to the homodu-
plices. When dsDNA of 100–1000 bp containing a mixture
of homo- and heteroduplices is electrophoresed through a
detection of unknown DNA polymorphisms. (A) Methods based on
homoduplices either by variations in electrophoretic mobility
natively, heteroduplices are cleaved by a mismatch specific endonuclease
epair Detection is based on recognition and repair of mismatches in a
llele sequences. (C) Mutation detection by MALDI-TOF MS is based on the
mentation spectrum. Abbreviations: TGCE: temperature gradient capillary
uring high performance liquid chromatography; MALDI-TOF MS: matrix-
Vol. 3, No. 2 2006 Drug Discovery Today: Technologies | Genomic technologies
Table 1. Comparison Summary Table
Technology Temperature
gradient capillary
electrophoresis
Denaturing
high-performance
liquid
chromatography
Enzymatic cleavage
(CEL I; SurveyorTM)
Matrix-assisted laser
desorption/ionization
time-of-flight mass
spectrometry
Mismatch
repair detection
Company SpectruMedix Transgenomic Transgenomic Sequenom Affymetrix
Varian Methexis Genomics
Pros High-throughput: short assay
times and automated sample
plates delivery
Flexibility: temperature gradient
allows screening of different
fragments in one run
High multiplexing potential
Convertible to DNA sequencer
Long record in disease
gene screenings
Mutant enrichment
possible
(fragment collector)
Allows high pool ratios
Amplicon
sizes >1 kb possible
Cleavage pattern
indicates mutation
position
Simple hardware
requirements
Reveals nature of
mutation without
sequencing
Short assay times
High multiplexing
potential
Extreme
multiplexing capacity
Biological mutant
enrichment allows
very high pool ratios
Cons Automated mutation scoring
results occasionally ambiguous
(fragment dependent)
Long assay times High assay costs with
commercially available
enzyme
Enzymatic sample
preparation required
before analysis
Significant set
up work requiredLimited multiplexing
potential
Limited multiplexing
potential
4 separate
reactions/runs
per probe required
Currently
commercially
not available
Refs [5,2] [11,9] [20] [33,36] [28]
gradient of denaturing agent, domains of the fragment dis-
sociate according to their Tm leading to decreased electro-
phoretic mobility (Fig. 1A). Retardation is greatest at the
point of partial denaturation and alleles are separated owing
to their different thermodynamic stability. The denaturant
used in electrophoresis is either urea or formamide (denatur-
ing gradient gel electrophoresis, DGGE) or temperature (tem-
perature gradient gel electrophoresis, TGGE) or combinations
thereof (Table 1).
The basis for mutation detection using single-stranded (ss)
DNA in single-strand conformation polymorphism (SSCP) is
the principle that upon heat denaturing and rapid cooling
ssDNA fragments with unique secondary structures form due
to intramolecular base pairing. The conformation depends
solely on the base composition and a single nucleotide
exchange can change the structure, causing different
electrophoretic mobilities under nondenaturing conditions
[1].
To increase throughput, resolution and sensitivity,
SSCP, DGGE, TGGE and their variations have all been
converted to capillary electrophoresis systems [2]. All of
them have been successfully used in human disease gene
screens. Mutation detection sensitivity with these systems is
usually above 90% with reaching up to 100% [3,4]. However,
many of the procedures still require substantial protocol
optimization specific for the analyzed fragments to reach
ultimate sensitivity. Currently, the most flexible system
appears to be temperature gradient capillary electrophoresis
(TGCE) where either a spatial or temporal temperature
gradient is used for allele separation [2]. A commercially
available TGCE system is supplied by SpectruMedix Corp.
(http://www.spectrumedix.com/) and the instrument is
designed for automated delivery of sample plates sets which
can be analyzed unattended [5]. Commercially available DNA
sequencer instruments (MegaBACE, GE Healthcare Bio-
Sciences, http://www.amershambiosciences.com; ABI Prism1
310, Applied Biosystems, http://www.appliedbiosystems.com)
have been used as well for a combined temperature/chemical
gradient mutation screening [3,6]. All these systems allow
fragment multiplexing with typical fragment sizes up to
700 bp. With reported detection limits of 5% mutant allele
frequency [5] analysis of pooled samples is a further option to
increase screening capacity to over 10,000 fragments per day
on a 192-capillary unit.
Future developments in conformation sensitive mutation
detection methods might be noted by reports from Li-Sucho-
leiki and colleagues, where the group succeeded in detecting
point mutations at frequencies of 10�6 in pooled human
genomic DNA [7]. Although the impressive detection sensi-
tivity of the system requires extensive processing work and
highly specialized procedures, the assay allows detection of
very rare alleles in large populations and a first automated
prototype system with similar detection sensitivity has been
described recently [8].
Denaturing high performance liquid chromatography
(DHPLC)
DHPLC is based on ion-pair reverse-phase chromatography,
which has long been used for HPLC size separation of DNA
fragments. Briefly, a separation cartridge contains a hydro-
phobic matrix and a mobile phase consisting of acetonitrile
and triethylammonium acetate buffer (TEAA). The positively
www.drugdiscoverytoday.com 125
Drug Discovery Today: Technologies | Genomic technologies Vol. 3, No. 2 2006
charged triethylammonium ion binds negatively charged
DNA and links the DNA with its hydrophobic groups to
the matrix. Application of an increasing acetonitrile gradient
releases the sample in order of increasing length. In DHPLC a
mixture of wild-type and mutant fragments containing
homo- and heteroduplices is applied to the cartridge at
partially denaturing temperatures. Binding of the heterodu-
plex molecules to the matrix is weaker due to temperature
induced helical distortion and the heteroduplices elute earlier
in the acetonitrile gradient [9]. Elution at temperatures that
induce partial heteroduplex denaturation is crucial for high
resolution and DHPLC adapted programs are available to
calculate the fragment specific temperature setting (http://
insertion.stanford.edu/melt.html) [10]. Typical fragment
sizes used in DHPLC mutation screens are in the range of
100–500 bp.
DHPLC has been extensively used for mutation analysis of
disease associated genes with detection sensitivities reaching
95–100% as well as for screening mutagenized animal model
libraries for genetic variants [11,12]. Detection limits down to
0.5% mutant allele frequency were achieved in a mitochon-
drial genome mutation screen [13].
DHPLC instruments specifically designed for mutation
scanning are currently commercially available from Transge-
nomic (http://www.transgenomic.com) and Varian (http://
www.varianinc.com). Both systems operate on 96-well- or
384-well-sample plate formats with automated sample deliv-
ery and assay times of about 4 min per sample. Working on
pooled samples can increase throughput whereas simulta-
neous analysis of multiplexed samples requires similar frag-
ment melting properties. Further improvements in
throughput can be achieved by multicapillary systems and
the introduction of laser-induced fluorescence detection in
combination with differential fluorophore fragment labeling
as described recently [14,15].
Endonuclease mismatch cleavage by CEL I
Recently, the plant endonuclease CEL I and the related
SurveyorTM nuclease (Transgenomic) have been used espe-
cially for the identification of chemically induced point
mutations from gene-driven large-scale mutagenesis screens
in a variety of organisms [16–18]. CEL I belongs to a family of
single-strand specific nucleases from plants and fungi and
detects single base mismatches and micro-insertions/dele-
tions of up to 12 bp in heteroduplex DNA [19,20].
For analysis, the sequence of interest with a size of up to
1.6 kb is amplified either from individual or pooled DNA and
heteroduplices are formed. Upon incubation with CEL I
heteroduplices are cleaved on the 30-side of the mismatch,
generating either single-stranded nicks and/or double-strand
breaks depending on the conditions applied (Fig. 1A). Diges-
tion products are analyzed either by, (i) agarose gel electro-
phoresis, (ii) denaturing polyacrylamide gel electrophoresis,
126 www.drugdiscoverytoday.com
(iii) fluorescent capillary electrophoresis or (iv) DHPLC.
Because the digestion products must add up in size to the
original amplicon length, false positive signals can be easily
excluded and the position of the mutation within the ana-
lyzed fragment might be pinpointed within a range of about
10 bp.
Reports from large-scale mutation screens describe CEL I
detection limits of 10% mutant allele frequency on standard
agarose gels and 6.25% for separation of fluorophore labeled
fragments via denaturing polyacrylamide gel electrophoresis
[16,18,21]. In addition, detection of 1 mutant allele in 16
copies (6.25% mutant allele frequency) or in 32 copies (3.1%
mutant allele frequency) has been described recently by
utilizing fluorescent capillary electrophoresis and DHPLC,
respectively [20]. For high-throughput demands, CEL I assays
on fluorophore labeled fragments generated from sample
pools allow mutation screening of several hundred kilobase
pairs on a single 96-well electrophoresis run by utilizing a LI-
COR DNA analysis system (LI-COR Biosciences, http://
www.licor.com) [17].
Future developments such as linker ligation to the cleaved
fragments for subsequent PCR cleavage product amplification
[22] or elimination of the interfering CEL I exonuclease
activity, responsible for removal of 50 labels and cleavage
product degradation, might increase CEL I mutation detec-
tion sensitivity and allow higher pool sizes. Inactivation of
the catalytic center, thus shifting CEL I to a mismatch bind-
ing protein for the enrichment of heteroduplices, could be
another strategy for CEL I based mutation detection on high
pool sizes [23].
Mismatch repair detection
ParAllele Bioscience, a company recently acquired by Affy-
metrix (http://www.affymetrix.com), has developed an ele-
gant mutation detection assay that is based upon the
biological detection of mismatched DNA in E. coli [24,25].
The principle behind the technology is that mismatches in
hemimethylated dsDNA activate the mismatch repair
mechanism in E. coli where a large portion of the unmethy-
lated strand is degraded and the double-strand is re-synthe-
sized using the methylated strand as a template [26].
Deletions or insertions larger than 5 bp do not trigger mis-
match repair [27] and the mismatch repair detection (MRD)
assay of ParAllele takes advantage of the E. coli mismatch
repair system to identify point mutations or micro deletions/
insertions.
For analysis, the sequence of interest is amplified by PCR
with amplicon sizes ranging from 150 to 500 bp [28]. PCR
amplicons are methylated in vitro and mixed with a methy-
lated linearized plasmid vector DNA carrying a marker gene
(e.g. lacZ) that was previously inactivated through the dele-
tion of an internal 5 bp fragment. In the next step a single-
stranded unmethylated standard DNA, consisting of the
Vol. 3, No. 2 2006 Drug Discovery Today: Technologies | Genomic technologies
corresponding wild-type amplicon and the full-length active
marker gene cloned into the same plasmid vector, is added.
Through denaturation, re-annealing and subsequent liga-
tion double-stranded hemimethylated circular plasmids,
which are composed of the methylated tester DNA with
an inactive marker gene on one strand and the unmethylated
reference DNA and the active marker gene by contrast, are
formed. The purified heteroduplex plasmids are then used to
transform E. coli, where mutation sorting takes place
(Fig. 1B).
If the test fragment does not contain any polymorphism,
the hybrid plasmid replicates without mismatch repair acti-
vation, generating two sorts of daughter plasmids with one
carrying the inactive and the other one carrying the active
marker gene. Thus, the presence of an active copy of the
marker allows identification of the cells on indicator media,
for example, formation of blue colonies. If the test fragment
does contain a polymorphism, mismatch repair degrades
large portions of the unmethylated strand including the
active marker gene and only plasmids carrying the inactive
marker gene are replicated. The cells are negative for the
marker protein and can be distinguished on indicator med-
ium, for example, by formation of white colonies.
The sensitivity of the system has been immensely
improved by introducing Cre recombinase in combination
with an episomal F0 factor carrying genes for tetracycline
resistance and streptomycin sensitivity flanked by loxP sites
[25]. As a result, cells transformed with a mismatch contain-
ing plasmid are rendered tetracycline resistant and can be
selected on respective media.
Although many enzymatic steps and hands on time are
required, the enormous power of MRD is its high potential for
multiplexing and pooling. In a recent report more than 1200
exons from a genomic region thought to contain genes
associated with autism were screened for polymorphisms
in a single MRD reaction and further expansion to 3000-plex
reactions seems feasible [28]. In addition, the assay can detect
mutant allele frequencies of 1% or less making sample pool-
ing another option for multiple target mutation screens on
large sample numbers [29].
Polymorphism detection using MALDI-TOF MS
Matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry is an attractive platform for high-through-
put SNP genotyping [30]. Recent developments extended the
application of MALDI-TOF MS for the scanning of unknown
mutations in target sequences with sizes up to 700 bp [31,32].
Briefly, the sequences of interest are PCR amplified using
primers that incorporate two different RNA polymerase pro-
moter sequences (e.g. T3, T7 or SP6) on each end of the
amplification product (Fig. 1C). Subsequently, RNA tran-
scripts are generated by in vitro transcription from the differ-
ent promoters and the transcripts are cleaved by nucleotide-
specific ribonucleases such as RNase A (C- and U-specific
cleavage) or RNase T1 (G-specific cleavage). Optionally, the
pyrimidine specific RNaseA can be directed to a C or U specific
cleavage through the substitution of rNTPs by noncleavable
dCTP or dTTP/dUTP nucleotides during in vitro transcription
using a modified RNA polymerase [33]. The cleavage products
are then separated by MALDI-TOF mass spectrometry to
generate an experimental mass signal pattern, where each
peak represents at least one cleavage fragment. Comparison
of the experimental cleavage pattern with the wild-type
fingerprint or with in silico reference mass signal patterns
reveals the presence of a mutation as mass shifts of peaks,
missing or additional peaks. Determination of the precise
mass of the additional or shifted peaks and calculation of the
nucleotide composition allows the identification of the
underlying sequence variation but requires that both strands
be analyzed.
Alternative methods have been developed where the in
vitro transcription is avoided by replacement of dTTP by dUTP
during PCR and subsequent treatment with Uracil-DNA gly-
cosylase to create abasic sites, which are sensitive for alkaline
cleavage [34]. Another nonenzymatic fragmentation strategy
comprises the chemical cleavage at acid-labile pyrimidine
nucleotides, incorporated through the replacement of dTTP
or dCTP by corresponding P30-N50-phosphoamidate nucleo-
bases during a primer extension reaction [35].
Detection accuracy of 95% or greater on three- to fivefold
multiplexed samples have been reported for MALDI-TOF MS
mutation screening [36]. A commercially available system
(MassARRAYTM) is supplied by Sequenom (http://www.
sequenom.com) in collaboration with Methexis Genomics
(http://www.methexis-genomics.com), which allows accord-
ing to the manufacturers specifications the analysis of up to 3
million bp per day.
Conclusion
The described procedures use different principles for detect-
ing novel sequence polymorphisms (summarized in Fig. 1)
and all have proven to be cost effective and reliable methods
for mutation screens. Nevertheless each of the methods has
characteristics, which could make it preferable for specific
applications. The flexibility of a temperature gradient for
allelic discrimination could favor TGGE over other methods
when a wide spectrum of target genes are screened for varia-
tions like in reverse genetic screens on animal model organ-
isms [37,38]. Under conditions, where run parameter are
optimized for the analyzed fragments, DHPLC shows excel-
lent detection rates and might be the method of choice for
screening large sample numbers on limited targets. CEL I
based mismatch cleavage, which allows mutation screening
on large PCR fragments, could find preferred application in
mutation screens on large target sequences such as cDNAs,
mono-exonic genes (like most G-protein coupled receptor
www.drugdiscoverytoday.com 127
Drug Discovery Today: Technologies | Genomic technologies Vol. 3, No. 2 2006
Outstanding issues
� Throughput and sensitivity of the available techniques are still
limiting factors for mutation screens of large genomic regions or
sample numbers.
� Increased throughput demands a higher degree of automation.
� Methods that simultaneously disclose the nature of the mutation are
preferable.
genes) or genomes with small intron sizes (C. elegans). In
addition, CEL I assays can be done generally with standard
laboratory equipment which renders the method extremely
cost efficient. MALDI-TOF MS has lately been developed for
mutation scanning and hence application reports are limited
but its multiplexing and automation potential makes it an
attractive tool for population screens. Finally, the biological
selection of mutant alleles in MRD offers unsurpassed multi-
plexing potential suitable for simultaneous multi-target
mutation screens on large patient cohorts. Unfortunately,
the system is not yet commercially available from Affymetrix.
Systematic investigations on mutation detection technol-
ogies for a side-by-side comparison of detection sensitivities
were lacking until recently [21]. The authors compared the
performance of DHPLC, MALDI-TOF MS and CEL I for muta-
tion detection on a set of pooled samples and the results
showed that all systems were able to identify the mutations
under appropriate conditions but CEL I was superior to detect
mutations in higher pooling ratios.
All procedures, except MALDI-TOF MS, finally require
sequencing of the mutant fragment to determine the nature
of the mutation and to exclude false positive signals, which
can occur in significant numbers. Double-stranded Sanger
DNA sequencing has long been considered as the gold stan-
dard for mutation detection but suffers from high assay costs
and low throughput. In addition, high-throughput sequen-
cing generates an enormous amount of data sets, which
requires powerful software tools for polymorphism detection
especially in screens for heterozygous mutations or poly-
morphisms present at low frequency. The detection of
mutant alleles present at a frequency <20% can be difficult
by DNA sequencing which might raise problems for mutation
detection in genetically heterogeneous diseases [39] or het-
erogeneous samples, for example, tumor biopsies. Recent
developments in sequencing technologies appear to have
the potential to dramatically improve throughput at a frac-
tion of costs to allow personalized human genome sequen-
cing or sequencing of disease-associated loci in large cohorts
(for review see [40]). Whether such next generation sequen-
cing technologies will be competitive for mutation detection
remains to be seen. Until then the described procedures are
the methods of choice for high-throughput mutation detec-
tion at reasonable costs.
Related articles
Kristensen V.N. et al. (2001) High-throughput methods for detection
of genetic variation. Biotechniques 30, 318–322
Larsen, L.A. et al. (2001) Recent developments in high-throughput
mutation screening. Pharmacogenomics 2, 387–399
Twyman, R.W. (2004) SNP discovery and typing technologies for
pharmacogenomics. Curr. Top. Med. Chem. 4, 1423–1431
128 www.drugdiscoverytoday.com
References1 Orita, M. et al. (1989) Detection of polymorphisms of human DNA by gel
electrophoresis as single-strand conformation polymorphisms. Proc. Natl.
Acad. Sci. U S A 86, 2766–2770
2 Murphy, K.M. and Berg, K.D. (2003) Mutation and single nucleotide
polymorphism detection using temperature gradient capillary
electrophoresis. Expert Rev. Mol. Diagn. 3, 811–818
3 Bjorheim, J. et al. (2001) Automated constant denaturant capillary
electrophoresis applied for detection of KRAS exon 1 mutations.
Biotechniques 30, 972–975
4 Murphy, K. et al. (2003) Evaluation of temperature gradient capillary
electrophoresis for detection of the Factor V Leiden mutation: coincident
identification of a novel polymorphism in Factor V. Mol. Diagn. 7, 35–40
5 Li, Q. et al. (2002) Integrated platform for detection of DNA sequence
variants using capillary array electrophoresis. Electrophoresis 23, 1499–1511
6 Kristensen, A. et al. (2002) Detection of mutations in exon 8 of TP53 by
temperature gradient 96-capillary array electrophoresis. Biotechniques 33,
650–653
7 Li-Sucholeiki, X.C. et al. (1999) Applications of constant denaturant
capillary electrophoresis/high-fidelity polymerase chain reaction to
human genetic analysis. Electrophoresis 20, 1224–1232
8 Li, Q. et al. (2005) Design of an automated multicapillary instrument with
fraction collection for DNA mutation discovery by constant denaturant
capillary electrophoresis (CDCE). J. Sep. Sci. 28, 1375–1389
9 Xiao, W. and Oefner, P.J. (2001) Denaturing high-performance liquid
chromatography: a review. Hum. Mutat. 17, 439–474
10 Jones, A.C. et al. (1999) Optimal temperature selection for mutation
detection by denaturing HPLC and comparison to single-stranded
conformation polymorphism and heteroduplex analysis. Clin. Chem. 45,
1133–1140
11 Lilleberg, S.L. (2003) In-depth mutation and SNP discovery using DHPLC
gene scanning. Curr. Opin. Drug Discov. Dev. 6, 237–252
12 Quwailid, M.M. et al. (2004) A gene-driven ENU-based approach to
generating an allelic series in any gene. Mamm. Genome 15, 585–591
13 van Den Bosch, B.J. et al. (2000) Mutation analysis of the entire
mitochondrial genome using denaturing high performance liquid
chromatography. Nucleic Acids Res. 28, e89
14 Premstaller, A. et al. (2001) Temperature-modulated array
high-performance liquid chromatography. Genome Res. 11, 1944–1951
15 Xiao, W. et al. (2001) Multiplex capillary denaturing high-performance
liquid chromatography with laser-induced fluorescence detection.
Biotechniques 30, 1332–1338
16 Till, B.J. et al. (2003) Large-scale discovery of induced point mutations with
high-throughput TILLING. Genome Res. 13, 524–530
17 Wienholds, E. et al. (2003) Efficient target-selected mutagenesis in
zebrafish. Genome Res. 13, 2700–2707
18 Winkler, S. et al. (2005) Target-selected mutant screen by TILLING in
Drosophila. Genome Res. 15, 718–723
19 Oleykowski, C.A. et al. (1998) Mutation detection using a novel plant
endonuclease. Nucleic Acids Res. 26, 4597–4602
20 Qiu, P. et al. (2004) Mutation detection using Surveyor nuclease.
Biotechniques 36, 702–707
21 Greber, B. et al. (2005) Comparison of PCR-based mutation detection
methods and application for identification of mouse Sult1a1 mutant
embryonic stem cell clones using pooled templates. Hum. Mutat. 25,
483–490
Vol. 3, No. 2 2006 Drug Discovery Today: Technologies | Genomic technologies
22 Zhang, Y. et al. (2002) An amplification and ligation-based method to scan
for unknown mutations in DNA. Hum. Mutat. 20, 139–147
23 Yeung, A.T. et al. (2005) Enzymatic mutation detection technologies.
Biotechniques 38, 749–758
24 Faham, M. and Cox, D.R. (1995) A novel in vivo method to detect DNA
sequence variation. Genome Res. 5, 474–482
25 Faham, M. et al. (2001) Mismatch repair detection (MRD): high-
throughput scanning for DNA variations. Hum. Mol. Genet. 10, 1657–1664
26 Modrich, P. (1991) Mechanisms and biological effects of mismatch repair.
Annu. Rev. Genet. 25, 229–253
27 Parker, B.O. and Marinus, M.G. (1992) Repair of DNA heteroduplexes
containing small heterologous sequences in Escherichia coli. Proc. Natl.
Acad. Sci. U S A 89, 1730–1734
28 Faham, M. et al. (2005) Multiplexed variation scanning for 1,000
amplicons in hundreds of patients using mismatch repair detection (MRD)
on tag arrays. Proc. Natl. Acad. Sci. U S A 102, 14717–14722
29 Fakhrai-Rad, H. et al. (2004) SNP discovery in pooled samples with
mismatch repair detection. Genome Res. 14, 1404–1412
30 Tost, J. and Gut, I.G. (2005) Genotyping single nucleotide polymorphisms
by MALDI mass spectrometry in clinical applications. Clin. Biochem. 38,
335–350
31 Krebs, S. et al. (2003) RNaseCut: a MALDI mass spectrometry-based
method for SNP discovery. Nucleic Acids Res. 31, e37
32 Hartmer, R. et al. (2003) RNase T1 mediated base-specific cleavage and
MALDI-TOF MS for high-throughput comparative sequence analysis.
Nucleic Acids Res. 31, e47
33 Stanssens, P. et al. (2004) High-throughput MALDI-TOF discovery of
genomic sequence polymorphisms. Genome Res. 14, 126–133
34 von Wintzingerode, F. et al. (2002) Base-specific fragmentation of
amplified 16S rRNA genes analyzed by mass spectrometry: a tool for rapid
bacterial identification. Proc. Natl. Acad. Sci. U S A 99, 7039–7044
35 Smylie, K.J. et al. (2004) Analysis of sequence variations in several human
genes using phosphoramidite bond DNA fragmentation and chip-based
MALDI-TOF. Genome Res. 14, 134–141
36 Ehrich, M. et al. (2005) Multiplexed discovery of sequence polymorphisms
using base-specific cleavage and MALDI-TOF MS. Nucleic Acids Res. 33, e38
37 Augustin, M. et al. (2005) Efficient and fast targeted production of murine
models based on ENU mutagenesis. Mamm. Genome 16, 405–413
38 Michaud, E.J. et al. (2005) Efficient gene-driven germ-line point
mutagenesis of C57BL/6J mice. BMC Genomics 6, 164
39 Takashima, H. et al. (2001) Screening for mutations in a genetically
heterogeneous disorder: DHPLC versus DNA sequence for mutation
detection in multiple genes causing Charcot-Marie-Tooth neuropathy.
Genet. Med. 3, 335–342
40 Jarvie, T. (2005) Next generation sequencing technologies. Drug Discov.
Today: Technol. 2, 255–260
www.drugdiscoverytoday.com 129