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Review |
Department of Clinical Chemistry, Medical Clinic, University Hospital Eppendorf, Martinistr. 52, D-20251 Hamburg, Germany.
a Author for correspondence. Fax +494047174621; e-mail wagener{at}uke.uni-hamburg.de
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Abstract |
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 Top Abstract IntroductionScreening MethodsScreening Methods for Point...Detection of Known MutationsDetection of Known Mutations:...References |
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Key Words: indexing terms: alleles • electrophoresis • gene insertions • gene deletions • polymerase chain reaction
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Introduction |
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TopAbstractIntroductionScreening MethodsScreening Methods for Point...Detection of Known MutationsDetection of Known Mutations:...References |
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1) What type of nucleic acid is analyzed (DNA or RNA)?
2) What kind of specimen is analyzed (e.g., peripheral blood, bone marrow, tissues, secretions, excretions)?
3) Are the mutations to be detected known before analysis?
4) How large is the number of potential mutations to be detected?
5) Need each of the potential mutations be detected?
6) What is the ratio between wild-type and mutant alleles?
7) How reliable is the method to be used, and how far can it be standardized?
8) How does the test perform?
9) Is the test suited for routine diagnosis?
10) What kind of quality assessment can be achieved?
Here, different methods for the detection of point mutations and small deletions or insertions will be discussed on the basis of the above criteria (for simplification, we shall refer to point mutations only in the text, though in general, small deletions or insertions are detected equally well by the methods described). In general, PCR is either used for the generation of DNA fragments, or is part of the detection method. Screening methods for unknown mutations as well as methods for the detection of known mutations are included. Though DNA sequencing techniques will not be covered, we stress that DNA sequencing is considered the gold standard and remains the definitive procedure for the detection of mutations so far. For this reason, mutations assumed from the results of screening methods must be confirmed by DNA sequencing. Special attention will be paid to performance and quality assessment. We do not intend to present an in-depth review. For detailed information the reader is referred to some review articles (1)(2).
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Screening Methods |
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TopAbstractIntroductionScreening MethodsScreening Methods for Point...Detection of Known MutationsDetection of Known Mutations:...References |
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In general, target sequences are amplified by PCR before analysis. At present, Taq polymerase is widely used for amplification. The error rate of Taq polymerase is in the range of 10-4 to 10-5 per nucleotide and is strongly affected by the reaction conditions (e.g., concentrations of magnesium chloride and dNTPs, pH, and temperature). Depending on the method of choice, polymerase errors may contribute reasonably to unspecific background, limiting the level of detection, particularly in situations where few mutated alleles are analyzed in a great excess of wild-type alleles (for theoretical considerations see ref. 3). Though at low statistical probability, errors may be misinterpreted as mutations when analyses are performed with low numbers of starting templates (<100 molecules; (4)). If polymerase errors are critical, positive results should be confirmed by alternative techniques and, though not applicable to all methods, thermostable polymerases with higher fidelity (e.g., Pfu DNA polymerase) may improve results in particular applications.
denaturing gradient gel electrophoresis (dgge), temperature
gradient gel electrophoresis (tgge)1
Principle.
Double-stranded (ds) DNA is electrophoresed
through a gradient of increasing concentration of a denaturing agent
(urea or formamide) or of increasing temperature. With increasing
concentration of denaturant or temperature, domains in the DNA
dissociate according to their melting temperature
(Tm). DNA hybrids of 100–1000 bp contain 2–5
such domains, each melting at a distinct temperature. Dissociation of
strands in such domains results in a decrease in electrophoretic
mobility. A 1-bp difference between two ds DNA homoduplices can change
the Tm by 1 °C or more. Base mismatches in
heteroduplices lead to a significant destabilization of domains,
resulting in differences of Tm between
homoduplex and heteroduplex of up to 6 °C. For this reason,
heteroduplices between wild-type and mutant fragments are generally
used for the analysis of point mutations. Theoretical melting profiles
can be predicted by appropriate computer programs (5) (for
a detailed review on DGGE see ref. 6).
Modifications.
To increase the number of melting domains
to be analyzed, GC-rich sequences are attached to one of the PCR
primers (GC clamp). With GC clamps, significantly more mutations were
detected by DGGE (7)(8).
Fragment size.
Maximum fragment size suited for DGGE is
~1000 bp. With increasing number of melting domains, the mobility
shifts decrease. For this reason, the fraction of mutations detected
decreases with increasing fragment size. In addition, time of
separation varies from 7.5 h to 10 h for fragment sizes in
the range of 50 to 1000 bp.
Detectable mutations.
According to data from the
literature and our own experiences, close to 100% of point mutations
can be detected when heteroduplices are generated from sense and
antisense strands and when GC clamps are attached
(6)(8)(9).
Detection limit.
DGGE or TGGE appears not to be suited
for the detection of a few mutant alleles in great excess of wild-type
alleles, since preselection of mutant alleles is not feasible.
Detection methods.
In the original report, radioactive
labeling of DNA fragments was performed (10). Radioactive
labeling has been replaced by ethidium bromide or silver stain.
Performance and quality assessment.
Before analysis,
optimal conditions for DGGE or TGGE must be determined either by
calculation on the basis of appropriate algorithms or by experimental
perpendicular gradient gel electrophoresis. In general, optimal
separation is evaluated experimentally. For both DGGE and TGGE, special
equipment is commercially available.
In principle, four bands are detectable in a heterozygous state after denaturation and renaturation corresponding to two homodimers (WW', MM') and two heterodimers (WM', W'M). With homozygous germline mutations, four bands are detectable only after the addition of wild-type DNA. The relative intensities of bands depend on the quantitative relation of mutant to wild-type DNA. This can pose difficulties, especially in solid tumors with variable amounts of nontumor DNA.
single-strand conformation polymorphism (sscp)
Principle.
Under certain conditions, single-stranded
(ss) nucleic acids form secondary structures in solution. The secondary
structure depends on the base composition and may be altered by a
single nucleotide exchange, causing differences in electrophoretic
mobility under nondenaturing conditions (11).
Modifications.
Initially, SSCP was described for the
analysis of DNA; however, analysis of RNA is also possible
(12)(13). Distinct secondary structures are
formed more frequently by RNA than by DNA molecules. In comparison with
DNA-SSCP, an additional step of in vitro transcription is required to
generate RNA from PCR fragments (13). With RNA, larger
fragments can be analyzed (13). Screening of multiple
fragments can be achieved by either restriction digest of larger PCR
fragments (14) or multiplex PCR
(15)(16). To identify potential mutations,
SSCP has been combined with direct DNA sequencing (17). In
several applications, minigels have been used instead of standard
sequencing gels (18)(19). However, whether the
resolution in a small gel is as high as in sequencing gels has not been
established. In other previous studies, mobility differences have been
analyzed by capillary electrophoresis instead of gel electrophoresis
(20).
Fragment size.
For optimal results, fragment size should
be in the range of 150 to 200 bp (21). The number of
detectable mutations decreases when larger fragments are analyzed. For
larger fragments, acceptable sensitivities may be achieved by RNA-SSCP
(13).
Detectable mutations.
Under optimal conditions (fragment
size <200 bp), ~80–90% of potential base exchanges are detectable
by SSCP (21). In addition to the size of the fragment,
assay performance is dependent on the concentration of glycerol within
the gel and the constancy of temperature during gel electrophoresis.
Except for G to T transversions, there appears to be no significant
effect of the type of base exchange on sensitivity (21).
In comparison with DNA-SSCP, higher sensitivities, especially for
analysis of fragments of >200 bp, have been reported for RNA-SSCP
(13).
Limit of detection.
Approximately one mutant cell is
detectable in the presence of 10 normal cells (22).
Methods of detection.
Initially, fragments were labeled
radioactively and detected by autoradiography. Nonradioactive
detection, e.g., by silver staining, is feasible.
Performance and quality assessment.
Electrophoretic
separation is carried out in nondenaturing polyacrylamide slab gels.
Depending on the polyacrylamide concentration, the size of the
fragment, and the presence of glycerol within the gel, time of
separation varies between 3 and 6 h. For higher resolution,
special gel matrices are commercially available (23).
Heating of gels during gel electrophoresis must be avoided. Adequate
convection of air is obligatory. Otherwise, no specialized equipment is
needed. Composition of reagents (e.g., concentration of glycerol) and
conditions of electrophoresis (e.g., concentration of acrylamide or
time of electrophoresis) are dependent on the characteristics of the
DNA fragments to be analyzed. Analysis of one fragment under different
conditions may increase the rate of detectable mutations. Optimal
conditions are largely determined empirically. Thus, standardization is
limited.
When SSCP is analyzed by gel electrophoresis, differences in mobility are evaluated by visual inspection. Standardization is limited in this setting. Similarly, automation is difficult to achieve. With the use of capillary electrophoresis instead of gel electrophoresis, both standardization and automation may be improved and the turnaround time will be decreased.
heteroduplex analysis (het)
Principle.
Heteroduplices are generated by heat
denaturation and reannealing of a mixture of wild-type and mutant DNA
molecules. In nondenaturing polyacrylamide gels, homoduplices and
heteroduplices exhibit distinct electrophoretic mobilities.
Modifications.
For higher resolution, special gel
matrices (MDE) can be applied instead of polyacrylamide gels
(23). Sharpening of bands may be obtained by the
separation of duplices in the presence of 15% urea. When ratios of
mutant to wild-type alleles are undefined, wild-type DNA has to be
added to the sample, allowing sufficient formation of heteroduplices
for the detection of mutations (24).
Size of fragment.
The optimal fragment length for the
detection of point mutations varies between 200 and 600 bp; the
detection of mutations in PCR fragments of up to 900 bp has been
reported (25)(26).
Detectable mutations.
Though the method is widely used
for screening purposes, relatively few systematic studies on the
fraction of mutations detectable have been published. The detection of
mutations is mainly dependent on the position of the mismatch within
the DNA fragment and the type of mismatch. White et al.
(27) described the detection of eight of nine different
mutations by application of heteroduplex analysis. In one report, all
p53 mutations investigated by SSCP were likewise detectable
by heteroduplex analysis (23). The proportion of point
mutations detected by HET has been estimated to ~80%
(1).
Limit of detection.
Systematic studies are lacking. The
detection limit depends both on the relative signal intensity and the
separation of heteroduplex vs homoduplex. Ratios of mutant to wild-type
DNA of <1:5 may not be detectable (own observations).
Detection method.
Homo- and heteroduplices are detected
either by ethidium bromide or silver staining after gel
electrophoresis.
Performance and quality assessment.
Electrophoretic
separation is carried out in nondenaturing polyacrylamide sequencing
gels. Depending on fragment size, time for electrophoretic separation
varies between 14 and 30 h. So far, the method is not suitable for
automation but may be performed automatically in the future with
drastically reduced times of electrophoretic separation by application
of capillary electrophoresis (28).
rnase a cleavage method
Principle.
Under defined conditions, mismatches within
RNA:RNA or RNA:DNA heteroduplices are cleaved by RNase A. After
cleavage, labeled fragments are analyzed by denaturing gel
electrophoresis.
Detectable mutations.
Mutations of purine bases are
cleaved with low efficiency or remain uncleaved. For this reason, by
analysis of RNA:DNA heteroduplices, only 30% to 40% of the possible
mutations are detectable. When both DNA sense and antisense strands are
analyzed, detection rate can be increased up to 70% (29).
Limit of detection.
No detailed information is available
with respect to the maximum ratio of mutant to wild-type alleles at
which the detection of mutant alleles is still possible.
Fragment size.
The maximum size of RNA that can be
analyzed is ~1000 bp. Analysis of larger fragments results in high
background due to unspecific cleavage at sites of perfect base pairing.
Incomplete separation of RNA:DNA duplices may occur when large
fragments are separated under denaturing conditions, making
interpretation of results difficult (6).
Comment.
Since only 70% of all types of mutations are
detectable, the method appears not to be suited for screening purposes
when compared with other methods. For this reason, aspects of routine
application and quality assessment will not be discussed in detail.
chemical cleavage method (ccm)
Principle.
Mispaired nucleotides within heteroduplices
are modified by chemical agents by using Maxam–Gilbert sequencing
chemistry. Hydroxylamine reacts with mispaired cytosine residues,
osmium tetroxide with mispaired thymine residues. DNA:DNA or DNA:RNA
heteroduplices are cleaved by piperidine at the sites of chemical
modification. If sense and antisense strands are analyzed, all point
mutations will be detected. Unspecific cleavage of homoduplices does
not present a problem when the method is performed appropriately
(30).
Modifications.
Originally, the method was described for
the analysis of DNA:DNA heteroduplices, but it may also be applied for
the analysis of DNA:RNA heteroduplices (1). When low
amounts of mutant alleles are analyzed in a large background of
wild-type DNA, sensitivity can be increased by separation and detection
of fluorescence-labeled fragments on a DNA sequencer (31).
Detectable mutations.
In principle, all possible
mutations are detectable by CCM (1)(30). It
has been reported that certain T:G mismatches are not modified by
osmium tetroxide. However, when both sense and antisense strands are
analyzed, a reliable detection of all types of point mutations is
achieved.
Detection limit.
By application of fluorescence
labeling, down to one mutant cell was detectable in a background of 10
nonmutated cells when separation and detection of fragments was
performed on a DNA sequencer apparatus (31).
Fragment length.
As outlined by Cotton (1),
DNA fragments of up to 2 kb can be analyzed by CCM.
Methods of detection.
Cleaved fragments are analyzed by
gel electrophoresis. Originally, 32P-end-labeled fragments
were used. Improved resolution of signals is obtained by labeling with
35S (32). In addition, silver staining may be
applied for detection (33). Fluorescence labeling was
mentioned above (31).
Performance and quality assessment.
The major
disadvantage of the CCM is the use of toxic substances. Because several
steps of the reaction must be carried out under a fume hood, the
potential for automation is limited. The major advantages of the CCM is
the fact that all point mutations are detected when sense and antisense
strands are analyzed. Furthermore, an objective measurement of reaction
products is feasible. Thus, separation and detection of DNA fragments
may be standardized by the application of fluorescence-labeled primers
in conjunction with a DNA sequencer (31) or possibly by
capillary electrophoresis in the future.
enzyme mismatch cleavage (emc)
Principle.
Heteroduplices generated by heat denaturation
and renaturation of PCR products of polymorphic DNA or wild-type and
mutant alleles, respectively, are incubated and cleaved by either the
bacteriophage T4 endonuclease VII or T7 endonuclease I (bacteriophage
resolvases). Subsequently, DNA fragments are analyzed by gel
electrophoresis (34)(35).
Fragment size.
Mutations were detectable in PCR products
between 88 and 940 bp (34) or up to 1.5 kb
(35).
Detectable mutations.
By application of both enzymes in
parallel, cleavage of heteroduplices was observed with all types of
small deletions (1- and 3-bp deletions in the APC gene or
the CFTR gene, respectively) and 13 of 14 point mutations
representing all types of possible nucleotide exchanges. By application
of only one enzyme, 11 of 14 mutations were identified. Although G to A
transitions were detectable in most cases analyzed, the G to A exchange
of the G551D mutation in the CFTR gene remained undetectable
even when both enzymes were applied (34). In a second
report, 17 of 18 point mutations and 3 of 4 small deletions were
detectable by application of T4 endonuclease VII only
(35). Both reports observed nonspecific background bands
of undetermined origin.
Limit of detection.
So far, bacteriophage resolvases
have been applied to the analysis of heterozygous states. No systematic
studies regarding the least ratio of mutant to wild-type alleles
detectable are known to us.
Method of detection.
In both reports mentioned above
(34)(35), 32P-labeled primers were
used for PCR. Subsequently, products were incubated with resolvases,
separated on polyacrylamide gels under nondenaturing or denaturing
conditions, and fragments detected by autoradiography. Silver staining
should be feasible.
Performance and quality assessment.
A number of problems
have to be solved before EMC can be considered as a routine screening
method for mutations. With the enzyme preparations used so far,
unspecific cleavage of homoduplices has been observed. The use of
highly purified enzymes may solve this problem. Since homozygous mutant
samples should not generate a specific signal, wild-type DNA has to be
added to detect these mutations. Some mutations are poorly recognized
by resolvases, resulting in digestion of only a small fraction of the
DNA. It has been suggested that mutant resolvases may be developed that
tightly bind a mismatch, but fail to cut it. This would allow the
detection of mutations in a solid-phase assay (36). Since
unspecific cleavage of homoduplices may occur, homoduplices must be
included as internal negative controls. The occurrence of unspecific
bands may pose a problem for the correct interpretation of results.
Additional experience with the use of resolvases is required before
detailed suggestions on quality assessment can be given.
cleavase fragment length polymorphism (cflp)
Principle.
CFLP analysis is a relatively recent method
(37). When single strands of DNA refold after
denaturation, sequence-dependent secondary structures consisting of
folded, hairpin-like configurations are formed. The cleavase I
endonuclease cleaves just 5' of the hairpin loop at the junction
between ss and ds DNA, generating a collection of fragments that is
unique to that strand of DNA. Changes in the sequence (e.g., single
point mutations, insertions, or deletions) of that strand will alter
the secondary structures formed and the CFLP pattern detected.
Fragment size.
Fragments of up to 2 kb can be analyzed.
Detectable mutations.
No systematic studies are
available yet.
Limit of detection.
No systematic studies are available
yet.
Method of detection.
CFLP patterns are resolved on
short, denaturing polyacrylamide gels. Detection of fragments proceeds
through labeling (e.g., biotin, 32P) of one of the PCR
primers.
Performance and quality assessment.
Automation may be
feasible by use of fluorescein-labeled primers in conjunction with
capillary electrophoresis. As a control, wild-type alleles must be run
in parallel. Additional experience is needed for detailed suggestions
regarding quality assessment.
mutation detection by mismatch binding proteins
Principle.
Mutations are detected by binding of the MutS
protein, a component of the Escherichia coli DNA mismatch
repair system, to ds DNA molecules containing mismatched bases
(38).
Modifications.
Heteroduplices were generated by heat
denaturation and subsequent reannealing after PCR amplification of
wild-type and mutant alleles. DNA duplices were incubated with the MutS
protein, and mutations were detected by mobility shift assays
(38). A simple assay is based on the fact that MutS
binding protects heteroduplex DNA from exonuclease digestion
(39). It has been suggested that a solid-phase assay may
be feasible, in which immobilized MutS binds mismatches in
heteroduplices.
Detectable mutations.
MutS binds different mismatches
with different affinities, and some mismatches (e.g., A:C) are bound
poorly (40). According to Lishansky et al.
(38), MutS bound more strongly to CFTR gene
heteroduplices containing the 
F508 3-bp deletion in one
of the strands than to heteroduplices with a single base-pair mismatch.
The G542X and G551D point mutations of the CFTR gene were
successfully detectable by the MutS assays (38). In the
MutS protection assay, three G-A and a C-T exchange were detected
(39). Recently, a solid-phase assay version with
immobilized MutS has been described (41).
Limit of detection.
Until now, only heterozygous
situations have been investigated. Systematic analysis of the detection
of mutations in a high background of wild-type DNA are lacking.
Methods of detection.
In the report by Lishansky et al.
(38), mutations were detected by gel mobility shift assays
with 32P-labeled PCR products. In the MutS protection
assay, fluorescence-labeled fragments were detected after separation on
a polyacrylamide sequencing gel (39).
Performance and quality assessment.
Reliability of the
assay depends primarily on the discrimination of MutS binding to
heteroduplices against the background binding to homoduplices.
Unspecific signals have been reported for homoduplices consisting of
either wild-type alleles or F508 alleles. However,
differences in signal intensities between homo- and heteroduplices
allowed the correct interpretation (38). In the same
report, a lower stability of the DNA–protein complex and decreased
signal-to-noise ratios have been described for point mutations in
comparison with the F508 deletion in the CFTR
gene. Unspecific background binding has been speculated to result from
errors of the Taq polymerase. However, because unspecific
binding of MutS to homoduplices did not increase with fragment length
and because the use of a polymerase of higher fidelity (Pfu
polymerase) did not reduce unspecific binding, this assumption has not
yet been verified (38).
Recently, the human mismatch binding factor has been shown to consist of two proteins, the MutS homolog hMSH2 and a second protein, GTBP (42). Since both proteins are required for mismatch binding, in vitro detection of mismatches may be improved by the use of both proteins. It has been suggested that artificial mismatch binding proteins may be generated from resolvases by site-directed mutagenesis. These mutant resolvases would tightly bind a mismatch, but fail to cut it (36). Possibly, the use of different mismatch binding proteins may allow the detection of mutations in a simple solid-phase assay format in the future.
So far, several problems must be solved before the detection of mismatches in heteroduplices by binding proteins can be applied as a screening method for point mutations. For this reason, considerations regarding standardization and quality assessment appear premature.
protein truncation test (ptt)
Principle.
The PTT is based on a combination of PCR,
transcription, and translation. The test selectively detects
translation-terminating mutations, which are revealed on the protein
level by sodium dodecyl sulfate (SDS)–polyacrylamide gel
electrophoresis (PAGE). A T7 promoter and an eukaryotic translation
initiation sequence is linked to a PCR primer
(43)(44). Subsequently, PCR products are used
as templates in coupled transcription–translation reactions. The size
of translation products is analyzed by gel electrophoresis. Stop codons
generated by point or frameshift mutations lead to a premature stop of
translation and to a reduced size of the translated proteins.
Modifications.
The methodological approach may start
with reverse transcription of mRNA. The resulting cDNA is amplified by
PCR (RT-PCR). Gross rearrangements and mutations affecting splicing are
detectable already by analysis of the RT-PCR products
(43)(44). Alternatively, mutational hotspot
regions may be amplified from genomic DNA
(44)(45). The T7 promoter and an eukaryotic
translation initiation sequence may be linked to a primer used for only
one round of PCR amplification (44)(45) or to
nested primers in a second round of amplification (43).
Chain-terminating mutations have also been detected by cloning of DNA
fragments in-frame with a colorimetric marker gene (lacZ)
followed by screening for the level of functional activity of the
marker polypeptide (ß-galactosidase) (46).
Size of fragments.
Sequences as long as 4–5 kb generate
enough PCR product to allow their subsequent transcription and
translation (43). The upper size limit seems to depend on
the ability to generate sufficient full-size RT-PCR product and on the
difference between the size of wild-type and truncated proteins.
Detectable mutations.
The assay detects
translation-terminating mutations generated by either point or
frameshift mutations. Missense mutations are not detected.
Polymorphisms or silent mutations do not pose a problem in this assay.
The method is particularly well-suited for genes in which
translation-terminating mutations dominate, e.g., for the analysis of
the APC and BRCA1 tumor suppressor genes in which
>80% of mutations result in truncated proteins
(44)(47) or the NF1
(neurofibromatosis 1) gene in which PTT detected close to 70% of the
mutations (48).
Detection limit.
No report is known to us in which the
minimum ratio of mutant to wild-type alleles has been analyzed.
Detection methods.
In vitro translation has been
performed in the presence of radioactively labeled amino acids. After
electrophoresis, protein fragments were detected by autoradiography. In
the cloning assay in which ß-galactosidase was used as a marker,
recombinant colonies were screened with blue/white color selection.
Performance and quality assessment.
No special equipment
beyond the standard equipment of a molecular genetic laboratory is
needed.
Translation-terminating mutations close to the 5' primer binding site are expected to result in very short translation products that might escape detection. Alternatively, if the truncating mutation is located near the binding site of the 3' primer, the lengths of the truncated and the wild-type translation products might be so close that the two peptides cannot be resolved by SDS-PAGE analysis (45). Additional bands, probably representing isoforms, e.g., due to alternative splicing or technical artifacts, may complicate the interpretation of results. So far, differences in electrophoretic mobility of truncated vs nontruncated proteins have been analyzed by visual inspection. In the future, proteins may be separated by chromatography or capillary electrophoresis, allowing an objective measurement of truncated proteins. Since PTT involves a number of steps (RT-PCR, in-vitro transcription and translation, gel electrophoresis), internal positive controls should be included.
allele-specific oligonucleotide (aso) hybridization on dna chips
Principle.
In DNA chips, the principle of ASO
hybridization has been extended to a screening method for mutations
(for recent reviews see refs. 49 and 50). This
has been made possible by the fabrication of high-density
oligonucleotide arrays. Oligonucleotides of known sequences are
immobilized on appropriate surfaces. Given a consensus sequence, a set
of four probes can be defined for each nucleotide in the target. Thus,
to screen 1000 nucleotides for a mutation or polymorphism would require
4000 probes. Labeled target sequences are hybridized to the immobilized
oligonucleotides. Because of their high resolution, fluorescent dyes
are best suited. In a commercial system (Affymetrix, Santa Clara, CA),
hybridization of the targets to the array is detected by
epifluorescence confocal scanning microscopy.
Modifications.
As solid supports, surface-modified
glass, polypropylene, or glass with small patches of polyacrylamide
have been described (50). Arrays of oligonucleotides
representing the complements of a known sequence are synthesized by
using combinatorial methods. In the Affymetrix system,
photolithographic methods are used in conjunction with nucleotide
precursors with a photocleavable protecting group
(51)(52). In the first report on the
application for screening polymorphisms of the HIV-1 clade B protease
(pr) gene, HIV-1 DNA or RNA was converted to a ds DNA
amplicon containing T3 and T7 RNA promoter sequences. The PCR amplicon
was transcribed with T7 or T3 RNA polymerase in the presence of
fluorescein-labeled rUTP. Fluorescein-labeled RNA was fragmented by
heating, hybridized to the chip, and analyzed (53).
Size of fragments.
The size of the DNA or RNA fragment
to be tested depends on number, size, and sequence of probes on the
array. In the first application published, a 382-bp contiguous region
of the HIV genome (pr gene) was analyzed by a high-density
array of 1.28 x 1.28 cm glass surface consisting of 12.224
different oligonucleotide probes (53).
Detectable mutations.
The pr chip mentioned
above was capable of determining 98.1% and 99%, respectively, of the
sense and antisense strands of four HIV-1 strains. When the sequences
from both strands were analyzed, all of the 382 bases were correctly
determined (53). In high-density oligonucleotide arrays,
multiple mutations occurring proximal to one another can lead to
noncalls or ambiguous calls.
Detection limit.
So far, no information is available
regarding the minimum ratio of mutant to wild-type alleles detectable
by high-density oligonucleotide arrays.
Detection methods.
For high-density arrays, fluorescence
has major advantages over other labeling procedures. Multiple colors
can be used to label different sequences, resolution is high, and
real-time measurements can be done. Both kinetic and equilibrium data
can be collected.
Performance and quality assessment.
For each target
sequence, a special chip containing appropriate oligonucleotides has to
be fabricated. Furthermore, a GeneChip-Scanner (Affymetrix) detection
instrument is needed. Because of limited experience, no definite
suggestions regarding quality assessment can be given at present.
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Screening Methods for Point Mutations and Small Deletions or Insertions: Summary and Conclusions |
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TopAbstractIntroductionScreening MethodsScreening Methods for Point...Detection of Known MutationsDetection of Known Mutations:...References |
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SSCP and HET are the most simple among the screening tests. A major advantage of HET over other methods is that running conditions do not have to be optimized, as conditions are constant for the majority of fragments and time for optimal separation of different sized fragments can be predicted. For SSCP, screening should be performed under different running conditions to achieve a maximum sensitivity, making this method more time consuming and labor intensive. Both methods allow simultaneously the rapid screening of different fragments with variable sizes in a single lane (e.g., products derived by multiplex PCR or after restriction digest of large PCR products), making these techniques particularly useful when large regions of DNA have to be screened in many patients. The main disadvantages of both tests is the fact that not all mutations are being detected. In addition, the size of fragments to be analyzed is limited, especially in SSCP. Close to 100% of mutations may be detectable by combining both techniques. However, no systematic evaluations of this assumption is known to us.
It is now well established that denaturing or temperature gradient gel electrophoresis detects close to 100% of point mutations. Comparative studies prove that TGGE (9) or DGGE (54) detects a higher proportion of point mutations than SSCP. Thus, if the detection of close to 100% of point mutations is intended, DGGE or TGGE should be applied. However, probably because of the relative ease of setup of SSCP, to date, published studies involving this method outweigh DGGE and TGGE roughly fourfold. A major disadvantage of TGGE or DGGE is that running conditions must be defined for each PCR product before analysis. Thus, compared with HET and SSCP, screening of large genes with many exons will be very labor intensive and time consuming, making these methods inefficient for some applications. At present, all of the electrophoretic methods are evaluated by visual inspection, which makes standardization difficult. Capillary electrophoresis may allow a more objective measurement in the future. In the chemical cleavage method, all point mutations are detected and large fragments can be screened. Because a number of analytical steps are required and toxic chemicals are applied, reports on the application of CCM are not as abundant when compared with the above methods. Mismatch cleavage by resolvases holds promise for the future, since standardization and automation should be achieved with relative ease. A major advantage of the mismatch cleavage method is that the size of the cleaved product roughly indicates the localization of the mutation. However, more experience is needed with these methods. The application of mismatch binding proteins has a great appeal for a simple screening test. However, discrimination of hetero- and homoduplices is not satisfactory at present. The application of PTTs will be restricted to the screening of genes in which translation-terminating mutations are abundant.
For all of the screening methods discussed so far, sequencing is advisable not only for the confirmation of results but also to assess the pathological significance of a specific mutation. Moreover, without sequence confirmation, unknown polymorphisms not associated with disease or technical artifacts may be misinterpreted as mutations, leading to false interpretations. Compared with the sequencing methods used initially, improvements have been achieved by the introduction of semiautomated high-throughput sequencing systems. However, as fully automated sequencing systems are not available at the moment, semiautomated sequencing is still labor intensive, costs are high, and assay performance as well as interpretation of results needs specialized personnel.
Many of the problems may be solved in the future by the introduction of DNA chip technology, which makes possible the combined detection and identification of mutations. However, for many applications, appropriate chips may not be available within the next years. Thus, screening methods for point mutations and small deletions most probably will keep their place in the diagnostic laboratory for a reasonable amount of time.
A major caveat is the lack of methods that are suited for the screening of mutant alleles at low abundance, compared with the wild-type alleles. Such methods are urgently needed for promising applications, such as in tumor diagnosis for the detection of mutant oncogenes in feces and secretions.
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Detection of Known Mutations |
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naturally occurring or primer-mediated restriction fragment
analysis
Principle.
Restriction enzyme recognition sites in DNA,
differing because of allelic variation or altered by mutations, are
cleaved specifically by restriction endonucleases only when the perfect
restriction recognition sequence is present. Commonly, fragments of
various sizes are analyzed by gel electrophoresis (restriction fragment
length polymorphism, RFLP). In case restriction sites are not affected
by mutations, artificial restriction sites can be introduced into the
target DNA by application of mismatched primers for PCR
(55)(56).
Modifications.
The "mutant-enriched PCR" is the most
important modification of this technique. In the original report,
artificial, primer-mediated restriction sites were introduced in
wild-type DNA by application of mismatched primers localized in the
direct vicinity of possible sites of mutations. As a result of base
substitutions, uncleavable restriction sites are generated from mutant,
but not from wild-type, alleles. Subsequent to the first round of
amplification, wild-type DNA is eliminated by restriction digest. In
the second round of amplification applying the same primers or
heminested primers, ideally PCR products representing mutant alleles
only are amplified (57)(58). Mutant-enriched
PCR would also be feasible with natural restriction sites if present.
The transfer of restriction digests from microtiter plates to horizontal gels is greatly facilitated by the so-called "microtiter array diagonal gel electrophoresis" (MADGE) (59)(60).
Detectable mutations.
All types of mutations are
detectable, for which differences in naturally occurring or
primer-generated restriction recognition sequences are present in
distinct alleles or wild-type and mutant DNA, respectively.
Limit of detection.
By simple RFLP analysis, one mutant
cell may be detectable in 50 to 100 nonmutant cells (57).
The detection limit can be lowered significantly by application of the
"mutant-enriched PCR" (57). Repeated restriction
digestion and PCR enriched for mutant alleles reportedly allows the
detection of one mutant ras allele in 106 normal
alleles (61). According to our experience, mutant-enriched
PCR is well suited to screen for mutant K-ras alleles in the
stools of patients with colorectal cancer (62).
Methods of detection.
Generally, fragments are analyzed
by electrophoresis in agarose gels and ethidium bromide staining. Other
methods of detection (e.g., hybridization, immobilization of labeled
fragments) are less common.
Performance and quality assessment.
For RFLP analysis, a
specificity of 100% is achieved when appropriate restriction enzymes
are used. As quality controls, different allelic variants or wild-type
and mutant DNA must be included in each analysis. Recognition sequences
may be destroyed by errors of the Taq polymerase. In
general, errors due to misincorporations will become detectable only
when high numbers of PCR cycles and (or) sensitive detection methods
are used. In the mutant-enriched PCR, false-positive results will be
obtained when a critical number of cycles is exceeded in the second PCR
subsequent to the restriction (57). The method has to be
adjusted to conditions such that no false-positive results are obtained
when variable amounts and different proportions of wild-type and mutant
DNA are analyzed. Questionable results may be confirmed by repetition
of experiments and subsequent sequencing of PCR products.
aso
Principle.
Mispairing of a single nucleotide within a
hybrid of 20 bp results in a decrease of the Tm
of ~5–7.5 °C. This difference in melting temperatures is adequate
for the specific detection of single nucleotide exchanges in DNA by
oligonucleotides. Cross-hybridization to irrelevant DNA sequences is
avoided by oligonucleotides with a minimum size of 16 to 20 bp.
Hybridization with larger oligonucleotides does not increase
sensitivity, because differences in Tm due to
mispairing of nucleotides decreases with increasing fragment length.
Generally, one of the reaction partners is immobilized to a solid
support.
Modifications.
Originally, electrophoretically separated
restriction fragments were immobilized on membranes and discriminated
by oligonucleotide hybridization (63)(64). In
more recent applications, the target DNA, generally obtained by PCR,
was immobilized to membranes without gel electrophoresis (dot blot).
The original dot-blot method is laborious when different allelic
fragments (e.g., HLA locus) or various mutated fragments (e.g.,
CFTR gene) are used to probe immobilized target fragments.
Methodological improvement has been achieved by the reverse dot-blot
technique where different oligonucleotides are immobilized to the same
membrane, allowing the detection of different polymorphisms or
mutations in a single reaction (65). Further improvement
has been achieved by microtiter formats. For the detection of low
amounts of K-ras mutated cells in a large background of
nonmutated cells (e.g., for the detection of tumor cells in stools of
patients with colorectal or pancreatic carcinoma), DNA was amplified
and cloned. Subsequently, wild-type and mutant clones were
discriminated by ASO (66). In an electrophoretic variant,
hybrids of target sequences and labeled oligonucleotides were submitted
to electrophoresis in a horizontal 20% polyacrylamide gel at a
temperature gradient increasing with time. At the appropriate melting
temperature, the oligonucleotide was released. Thus, the freed rather
than the bound oligonucleotide is displayed. This technique has been
designated "profiling of oligonucleotide dissociation gel
electrophoresis" (PODGE) (67). Hybridization reactions
can also be performed in solution. For example, biotinylated primers
were used to amplify a fragment of the 
1-antitrypsin
gene containing a potential Z-mutation. Hybridization was performed in
solution with Eu-labeled matching or mismatch primers. After
immobilization on streptavidin-coated wells, mutations were detected by
washing at appropriate stringency (68).
ASO hybridization is the principle on which the design of DNA chips is based. DNA chips may be available in the near future, which make possible screening for a wide range of mutations and polymorphisms once these have been defined. In a recent variation of the chip technology, a contiguous stacking hybridization technique was applied for the detection of ß-thalassemia mutations (69).
Detection limit.
In reconstruction experiments, one cell
with a mutated ras gene was detectable in 10 cells with
wild-type alleles by the dot-blot technique (70). A large
increase in sensitivity was achieved by prior cloning of PCR fragments
and screening of individual clones with probes complementary to
different mutations of the K-ras gene. In this setting,
errors of the Taq polymerase may give rise to false-positive
results. Consequently, a cutoff of positive clones must be established
(66).
Detection methods.
Originally, 32P-labeled
probes were used for detection. For nonradioactive detection in direct
or reverse dot blots, avidin–peroxidase conjugate can be applied in
combination with biotinylated oligonucleotides or probes
(65). Detection of bound probes by time-resolved
fluorometry has been mentioned (68).
Performance and quality assessment.
The specificity of
ASO depends on accurate control of the hybridization conditions.
Because they depend on base sequence and particular base substitutions,
hybridization conditions must be defined precisely for each
application. In solid-phase applications, the effect of base
composition on the melting temperature can be minimized by the addition
of tetramethylammonium chloride during hybridization. In the
direct dot blot, signal intensity is influenced by the affinity of
the DNA to the membrane. Similarly, in the reverse dot blot, different
signal intensities may be obtained when multiple oligonucleotides are
immobilized to a different extent. To reach comparable signal
intensities, the concentrations of immobilized oligonucleotides must be
adjusted (65). Difficulties of interpretation may occur
when weak signals are obtained. For each analysis, matching and
mismatching controls have to be included for each allele or each
mutation, respectively. Samples and controls should be analyzed on the
same membrane when different samples are investigated in parallel.
Standardization is difficult when signal intensities are evaluated by
visual inspection. The use of microtiter plates, other surfaces, or
tubes in conjunction with devices to measure signal intensities of
bound hybrids appears to be better suited, both for automation and
standardization. In this context, the DNA chip technology represents a
major breakthrough. Many of the problems associated with conventional
solid-phase applications are avoided by the PODGE variant
(67).
allele-specific amplification (asa)
Principle.
PCR is performed in two parallel reactions.
In the first reaction, the 5' primer is complementary to the wild-type
sequence; in the second reaction, the 5' primer is complementary to the
mutant or polymorphic sequence. Assuming that elongation occurs only
when primer and target sequence match completely, only one allele of
either mutant or wild-type DNA is amplified. The method was developed
independently by different groups. Two different approaches have been
described in parallel. The first approach is based on the lack of
primer elongation due to a mismatch at the far 3'-end of the primer.
These methods have been named "amplification refractory mutation
system" (ARMS) (71), "allele-specific PCR" (ASPCR)
(72), "PCR amplification of specific alleles" (PASA)
(73), or ASA (74). In the second approach,
the mismatch is located within the primer, preventing primer annealing
when mispairing occurs. Methods based on this principle were called
"competitive oligonucleotide priming" (COP) (75) or
"color complementation assay" (CCA) (76).
Modifications.
Assuming a homozygous situation, lack of
amplification will occur in one of the reactions when PCR is performed
with different pairs of 5' primers, one complementary and the other not
complementary to the alleles. Internal controls must be included to
exclude false-negative results (e.g., for heterozygosity). By multiplex
PCR, developed, e.g., for the diagnosis of cystic fibrosis, a positive
signal is obtained in each reaction, circumventing this problem and
allowing the simultaneous detection of different alleles
(77). A further disadvantage of the original protocols is
the performance of two different reactions in parallel. Heterozygous or
homozygous status may be discriminated in a single reaction when
different alleles are amplified by primers labeled with different
fluorochromes (76)(78). In the ASA by
tetra-primed PCR, different alleles can be distinguished in a single
PCR, by using two annealing temperatures and four primers
(79).
Both pairs of primers elongating either one or the other allele can be applied in one reaction when the methodological variant "PCR amplification of multiple specific alleles (PAMSA)" is performed. One of the allele-specific primers carries an additional stretch of noncomplementary nucleotides at the 5'-end. Thus, amplification products of both alleles can be discriminated by differences in size (80)(81). A comparable method has been described as "double ARMS" (82).
Detectable mutations.
Depending on assay conditions and
mismatch, false extension of 3'-ends of primers may occur. Kwok et al.
(83) reported that yield of PCR products decreased by
100-fold for A:G, G:A, or C:C mispairing and by 20-fold for A:A
mispairing. Elongation of primers occurred in all other types of
mispairing. However, elongation of mismatched bases can be avoided when
appropriate primers and reaction conditions are applied. Specificity of
primer extension may be improved by appropriate adjustment of
experimental conditions (84)(85). Specificity
of the reaction is influenced by the concentration of magnesium,
primers, deoxyribonucleotides, target DNA, and Taq DNA
polymerase. Addition of formamide may reduce unspecific reactions.
Under optimized conditions all types of mismatches can be reproducibly
detected by ASPCR at comparable concentrations of different alleles or
wild-type and mutant DNA (84). Furthermore, a more
reliable inhibition of elongation is achieved by introduction of
additional mismatches 5' of the 3' end of the primer (77).
The specificity of the method is strongly influenced by the ratio of
mutant to wild-type DNA.
Limit of detection.
Identification of a homozygous or
heterozygous state is the main application of the ASA. This method is
reliable and flexible for the analysis of homozygous or heterozygous
states. Specificity is more critical when the ratio of mutant to
wild-type alleles is low and the actual fraction of mutant alleles is
unknown. The detection of few tumor cells carrying mutations of the
K-ras gene in the presence of a large number of normal cells
has been reported by several authors (86)(87)(88).
Methods of detection.
For most of the methods outlined
above, detection is performed by gel electrophoresis. Similar to other
applications, a main advantage of electrophoretic detection systems is
the possibility to control the appropriate size of fragments. By
application of fluorochrome-labeled primers, electrophoretic separation
is not necessary when primers in excess are removed before detection
(76).
Performance and quality assessment
The possibility of
false-positive or -negative results is the major limitation of ASA.
False-positive results may be due to contamination or imperfect
extension. Guidelines regarding the avoidance of contaminations should
be followed strictly. To exclude false results, reaction conditions
must be standardized and the concentration of target DNA must be
defined and controlled precisely. Target alleles should be included as
controls to exclude false-positive or false-negative results. Primers
complementary to alleles with and without mismatches should both be
used. Automation of the method by the use of solid supports and
nonradioactive detection systems is conceivable.
single nucleotide primer extension
Principle.
The principle is similar to that of ASA. The
method is based on the extension of the 3'-end of a primer by a single
labeled nucleotide. Extension occurs only when the labeled nucleotide
is complementary to the nucleotide of the target DNA adjacent to the
3'-end of the primer (89)(90). On the basis of
comparable fidelities, either T7 or Taq DNA polymerases can
be applied. Because of the high error rate, the Klenow fragment of
E. coli DNA polymerase is not suited (91). The
method is also known as "minisequencing."
Modifications.
In one of the first reports on this
method, two different reactions were performed with labeled nucleotides
either complementary to one allele or to the other allele
(90). In the second approach, specific nucleotides were
applied, leading to differences in the electrophoretic mobility of the
fragments (89). In addition, similar approaches, with
modifications mainly in the labeling strategies, have been reported by
several authors (91)(92). One modification is
based on the method of dideoxy sequencing. The 3'-end of the primer is
located upstream of the mutant nucleotide(s). Use of dideoxy
nucleotides complementary to the mutant nucleotide primer extension
will lead to a termination earlier in mutant alleles than in wild-type
alleles (93).
Specificity.
Reliable discrimination between different
alleles will be obtained when the reaction is performed under
appropriate conditions. Application of comparable amounts of different
DNA to be analyzed is required.
Detectable mutations.
All possible types of nucleotide
exchanges can be detected by the single nucleotide primer extension
method.
Detection limit.
No systematic analyses on the
performance of the technique at different ratios of wild-type to mutant
DNA are known to us.
Detection methods.
Nucleotides are either labeled by
32P (90), 3H, or by digoxigenin
(91). In general, products of primer extension are
analyzed by electrophoretic separation
(90)(92)(94). In one of the
original approaches, nucleotides were applied, modifying the
electrophoretic mobility of the fragments (89). The method
has been performed as a solid-phase technique, making automation
possible (91)(95).
Performance and quality assessment.
Because signals can
be quantified without electrophoresis, the method is well suitable for
automation. A nonradioactive automated solid-phase assay has been
described (91)(95).
Diagnosis is based on the comparison of the results with appropriate controls (homozygous, heterozygous, mutant vs wild-type). For reliable discrimination, minimal variation of positive signals and background is essential, making standardized amounts of DNA mandatory.
oligonucleotide ligation assay (ola)
Principle.
Two primers are hybridized to complementary
stretches of DNA at sites of possible polymorphisms or mutations;
primers are created such that the 3'-end of the first primer is located
immediately adjacent to the 5'-end of the second primer. Assuming that
the 3'-end of the first primer matches perfectly with the target DNA,
both primers can be ligated by DNA ligases (e.g., T4 DNA ligase). No
ligation will be obtained when a mismatch occurs at the 3'-end of the
first primer (96)(97).
Modifications.
In the original approach, ligated and
nonligated primers were discriminated by dot blot on the basis of
differences in hybridization conditions (96). A second
approach involved labeled primers where the first primer was
biotinylated at the 5'-end and the second primer was 32P-
or fluorochrome-labeled at the 3'-end. Differentiation of fragment size
by electrophoresis is feasible.
Detectable mutations.
All possible combinations of base
pairings between the 3'-end of the 5' primer and the target DNA have
been investigated. Under appropriate conditions, ligation will take
place only when the 3'-end of the primer matches perfectly with the
target sequences (97).
Limit of detection.
Studies on the specificity and
sensitivity of the method at variable ratios of mutant to wild-type DNA
are not known to us.
Performance and quality assessment.
Ligation of
complementary bases depends mainly on the concentration of salts and
the proportions of concentrations between ligase and DNA.
False-negative results will be obtained when high salt concentrations
or low concentrations of enzyme are applied (97). A low
variability of positive signals and background signals is essential for
reliable discrimination. Standardization of DNA extraction is
essential. Positive and negative controls should be included in each
assay.
An automated version of OLA has been described (98). For detection, the 5'-end of one primer was labeled with biotin and the 3'-end of the other primer was labeled with digoxigenin. After ligation and binding to streptavidin immobilized on the surface of a microtiter plate, digoxigenin was detected by an anti-digoxigenin antibody coupled to alkaline phosphatase catalyzing a substrate reaction.
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Detection of Known Mutations: Summary and Conclusions |
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TopAbstractIntroductionScreening MethodsScreening Methods for Point...Detection of Known MutationsDetection of Known Mutations:...References |
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Any one of the above methods is suited for the analysis of allelic differences in hereditary disease. Non-gel-based detection systems have been developed for most of the assays described, making these methods favorable for application in routine laboratories. For each technique, reaction conditions must be standardized and appropriate internal controls must be included. One must keep in mind that misleading results may be obtained because of unknown polymorphisms within the target region affecting, e.g., restriction enzyme recognition sequences or hybridization of probes and binding of primers.
In cases in which a large number of different mutations or polymorphisms are to be detected, the DNA chip technology most probably will be the method of choice in the near future. However, with a restricted set of mutations such as the factor V gene mutation in activated protein C resistance, methods that are technically less demanding will keep their place in clinical laboratories.
At present, primer-mediated restriction fragment analysis in conjunction with mutant-enriched PCR appears the technique best suited for the amplification of low-abundance mutated alleles in great excess of nonmutated alleles. In comparison with ASO hybridization after cloning of PCR fragments, primer-mediated restriction fragment analysis is technically simpler and sufficiently sensitive. So far, promising applications of the technique are the detection of K-ras mutations in stools (62) and bronchoalveolar lavage (99).
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Footnotes |
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References |
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TopAbstractIntroductionScreening MethodsScreening Methods for Point...Detection of Known MutationsDetection of Known Mutations:...References |
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1-antitrypsin deficiency. Clin Chem 1993;39:1626-1631.
[Abstract]
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M. S. Nauck, H. Nissen, M. M. Hoffmann, J. Herwig, C. R. Pullinger, M. Averna, J. Geisel, H. Wieland, and W. Marz Detection of mutations in the apolipoprotein CII gene by denaturing gradient gel electrophoresis. Identification of the splice site variant apolipoprotein CII-Hamburg in a patient with severe hypertriglyceridemia Clin. Chem., July 1, 1998; 44(7): 1388 - 1396. [Abstract] [Full Text] [PDF]
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B. S. Bharaj, K. Angelopoulou, and E. P. Diamandis Rapid sequencing of the p53 gene with a new automated DNA sequencer Clin. Chem., July 1, 1998; 44(7): 1397 - 1403. [Abstract] [Full Text] [PDF]
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J. R. Conejo, T. Parra, M. Cantero, A. Jimenez, V. Granizo, V. Gabriel de Arriba, and F. Carballo Detection of H-ras Mutations in Urine Sediments by a Mutant-enriched PCR Technique Clin. Chem., July 1, 1998; 44(7): 1570 - 1572. [Full Text] [PDF]
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 J. Korkko, S. Annunen, T. Pihlajamaa, D. J. Prockop, and L. Ala-Kokko Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing PNAS, February 17, 1998; 95(4): 1681 - 1685. [Abstract] [Full Text] [PDF]
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