current methods for high-throughput detection of novel dna polymorphisms

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 mobility
induced by conformational differences in single or double-

stranded DNA molecules.

� M
ethods based on DNA conformation induced retention
time differences in ion-pair reverse-phase chromatography

– denaturing high performance liquid chromatography

(DHPLC).

� M
ethods based on recognition and cleavage of mismatches
in HETERODUPLEX (see Glossary) molecules by specific endo-

nucleases and separation of the cleavage products.

� M
ethods based on recognition and repair of mismatches in
a biological system for selection and amplification of the

mutant allele sequence – mismatch repair detection (MRD).

� M
ethods based on nucleotide-specific enzymatic or
chemical 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

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http://dx.doi.org/10.1016/j.ddtec.2006.05.002

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

http://www.spectrumedix.com/

http://www.amershambiosciences.com/

http://www.appliedbiosystems.com/


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,


(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

http://insertion.stanford.edu/melt.html

http://insertion.stanford.edu/melt.html

http://www.transgenomic.com/

http://www.varianinc.com/

http://www.varianinc.com/

http://www.licor.com/

http://www.licor.com/

http://www.affymetrix.com/


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


http://www.sequenom.com/

http://www.sequenom.com/

http://www.methexis-genomics.com/


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


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