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Biochemistry and Biophysics |
Dana Farber Cancer Institute, Adult Oncology and Radiation Oncology [S. C., B. D. P., Y. Z., G. M., G. M. M.] and Molecular Diagnostics Laboratory [E. A. F.], Harvard Medical School, Boston, Massachusetts 02115, and University of California School of Dentistry, Los Angeles, California 90095-1668 [S. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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G) and wild-type DNA-fragment populations. Aldehydes are uniquely
introduced at the position of mismatched adenines via the
Escherichia coli glycosylase, MutY. Subsequent treatment
with a biotinylated hydroxylamine results in highly specific and
covalent biotinylation of the site of mismatch. For PCR amplification,
synthetic linkers are then ligated to the DNA fragments. Biotinylated
DNA is then isolated and PCR amplified. Mutation-containing DNA
fragments can subsequently be sequenced to identify type and position
of mutation. This method correctly detects a single TG transversion
introduced into a 7-kb plasmid containing full-length cDNA from the
p53 gene. In the presence of a high excess wild-type DNA
(1:1000 mutant:normal plasmids) or in the presence of diverse DNA
fragment sizes, the DNA fragments containing the mutation are readily
detectable and can be isolated and amplified. The present
Aldehyde-Linker-Based Ultrasensitive Mismatch Scanning has a current
limit of detection of one base substitution in 7 Mb of DNA and
increases the limit for unknown mutation scanning by two to three
orders of magnitude. Homozygous and heterozygous p53 regions (GT,
exon 4) from genomic DNA are also examined, and correct identification
of mutations is demonstrated. This method should allow large-scale
detection of genetic alterations in cancer samples without any
assumption as to the genes of interest. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Understanding the dependence of carcinogenesis on specific mutations is contingent on the availability of technologies that can sensitively and effectively screen cancer samples for unknown mutations in several fragments (genes) simultaneously. The ability to detect mutant alleles in the presence of an excess normal alleles, which often "contaminate" cancer samples, is another requirement that must be satisfied for screening mutations in cancer (8) . Although powerful methods are available to screen short sequences for known mutations (9) , most current methods for identifying unknown mutations remain limited to DNA fragments of a single size, typically <1 kb (10) , and require a high proportion of mutant DNA in the sample for adequate sensitivity.
We present a novel approach for mutation detection (Fig. 1
) that addresses several of the requirements described
above. The method combines: (a) the specificity of mismatch
repair glycosylases for faithful mismatch detection; (b) the
high selectivity of the biotin-avidin interaction for isolation of
biotinylated mutant DNA fragments; and (c) the sensitivity
of PCR to enable identification of the isolated mutant DNA. After
formation of heteroduplexes using "control" and "mutant" DNA
samples, mismatched adenines are excised via MutY glycosylase, in a
process that generates an unsaturated open chain aldehyde on the sugar,
at the position of the apyrimidinic site. A biotinylated hydroxylamine
(ARP3
) is then covalently reacted with the aldehyde, and the biotinylated DNA
is linker ligated and separated from nonbiotinylated DNA using
streptavidin-coated microspheres. The microspheres are then directly
used in a PCR reaction, which proceeds using the intact DNA strand that
is complementary to the biotinylated strand held on the microspheres.
Using this novel approach, isolation of mutated fragments does not
require prior knowledge of the sequence, can be performed in the
presence of 1000-fold excess normal alleles, and can be carried out for
large (
7 kb) and non-uniform DNA fragment sizes simultaneously.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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G
transversion, a serinealanine change) full-length human p53 sequence
(1843 bp) was produced via site-specific mutagenesis, via a
modification of the procedure described previously (11)
.
Briefly, human wild-type p53 cDNA was inserted into the
BamHI site of the expression vector pcDNA3.1(+) (Invitrogen,
CA). To generate the S378A mutation, p53 was subcloned into the
BamHI site of pAlter-1 and mutagenesis carried out using the
Altered Sites II mutagenesis system (Promega Corp., Madison, WI). The
sequence of the mutagenesis oligonucleotide was: GGG TCA GTC TAC CGC
CGC CCA TAA A. Mutations were conformed by sequencing, and the p53 cDNA
was then subcloned into pcDNA3.1(+).
Prior to heteroduplex formation, mixtures of mutant plasmid (mutp53)
and wild-type plasmid (wtp53) were formed at desired ratios in Sau3A1
buffer (10 mM NaCl, 1 mM Bis Tris Propane-HCl,
1 mM MgCl2, and 0.1 mM
DTT, pH 7.0), and samples were digested with Sau3A1 enzyme (New England
Biolabs) for 2 h at 37°C. Digestion lead to formation of 31
double-stranded fragments with a wide distribution of sizes up to
1000 bp, with AGCT ends. After digestion, the mixture was heated at
94°C for 2 min, gradually cooled down, and incubated for 1 h at
65°C and then allowed to cool slowly to room temperature. The sample
solutions were then treated with 30 mM freshly prepared
hydroxylamine (pH 7.0, for 1.5 h at room temperature) to remove
traces of spontaneously forming aldehydes in DNA (12, 13)
.
After incubation, the samples were phenol:chloroform extracted, ethanol
precipitated, and resuspended in the buffer of choice.
As a test system for chemiluminescence studies, 5' fluoresceinated
double-stranded oligonucleotides with or without a centrally located
A:T C:G base substitution were synthesized (Oligos Etc; (+)strand
sequence: fluorescein-5'-GTC TCC CAT CCA AGT ACT AAC
CAG GCC CGA CCC TGC TTG GCT TCC GAT T-3'). For
cross-hybridization of mutant and wild-type sequences, equimolar
amounts (0.3 µg) of each oligonucleotide were mixed in 40
mM Tris-HCl (pH 7.5), 20 mM
MgCl2, and 50 mM NaCl and then heated
at 94°C for 2 min and gradually cooled down and incubated for 1 h at 65°C to form heteroduplexes. The oligomers were then treated
with hydroxylamine as described above.
Genomic DNA from one patient known to harbor a heterozygous
mutation (first sample, GT transversion at codon 110, exon 4 of p53)
or from a second patient with no mutation (second sample), was used to
apply ALBUMS in screening genomic DNA. The presence or absence of the
mutations in exon 4 had been demonstrated via sequencing at the
Dana-Farber Cancer Institute Molecular Diagnostics Laboratory. To
amplify and purify exon 4 for ALBUMS-screening, PCR with a
high-fidelity polymerase (Advantage HF-2; Clontech) was used. The
20-mer primers used for PCR amplification of the 399-bp region were:
5'-CAA CGT TCT GGT AAG GAC AA-3' (forward) and 5'-GCC AGG CAT TGA AGT
CTC AT-3' (reverse). PCR thermocycling conditions were: 94°, 30 s; (94°, 20 s/65°, 20 s/68°, 20 s) x 10 cycles,
with annealing temperature decreasing 1°/cycle (touchdown PCR);
(94°, 10 s/55°, 20 s/68°, 20 s) x 25 cycles;
68°, 6 min; 4°; Hold. After amplification, the PCR product was
gel-purified to remove the small amount of genomic DNA (QIAquik gel
extraction kit; Qiagen, Inc.). Heteroduplex formation was performed as
described above for the plasmid system.
Verification of Biotinylated Aldehyde-reactive Probe Reactivity
with MutY-treated Heteroduplexes.
Heteroduplexes were resuspended in MutY buffer (10 mM
HEPES-KOH, 0.1 mM KCl, and 10 mM EDTA, pH 7.0)
prior to incubation with MutY (Trevigen) using 1 unit of enzyme/µg
DNA, at 37°C for 1 h. After MutY treatment, the sample was
incubated with 5 mM biotinylated ARP (14)
,
a biotinylated hydroxylamine, from Molecular Probes, for 30 min at room
temperature. Unreacted ARP was removed by microbiospin-6 column
(Bio-Rad) filtration, and the samples were fluoresceinated by a 1-h
exposure to a commercially available fluoresceination reagent
(Fluorescein Label IT reagent; 1 µl reagent/µg DNA in
4-morpholinepropanesulfonic acid buffer, pH 7.5, at 37°C). Excess
reagent was then removed by microbiospin filtration. In experiments
using the synthetic prefluoresceinated heteroduplexes, the
fluoresceination step was omitted. The doubly-labeled (ARP and
fluorescein) DNA fragments were then immobilized onto
neutravidin-coated microplates (Pierce) in the presence of 5
nM anti-fluorescein-Fab-alkaline phosphatase (Boehringer),
and chemiluminescence was performed using an Intensified Charge Couple
Device camera (Princeton Instruments) as described (13)
.
Experiments were repeated three to five times.
Generation of Random Aldehyde-containing DNA Fragments.
To test the ability of the method to isolate and amplify DNA
fragments that contain aldehydes, aldehyde-containing
apurinic/apyrimidinic sites were artificially introduced to DNA prior
to reaction with 5 mM ARP. For this purpose,
Sau3A1-digested wild-type plasmids were treated with hydroxylamine,
extracted in phenol-chloroform, and precipitated in ethanol. The
samples were then depurinated via exposure to sodium citrate (pH 3.5)
at 38°C for different time periods (15)
. The
depurination was stopped by raising the pH to 7.0. The reaction mixture
was then purified via microbiospin-6. Experiments were repeated three
times.
Isolation, PCR Amplification, and Sequencing of
Mutation-containing DNA.
After MutY treatment or acid depurination and exposure to ARP, the DNA
fragments were extracted in phenol-chloroform and precipitated in
ethanol. Samples were then resuspended in 60 µl of ligation buffer
for ligation of asymmetric linkers corresponding to Sau3A1-restriction
sites, following the protocol of Hubank and Schatz (16)
.
The linkers used were a 24-mer, 5'-AgCACTCTCCAgCCTCTCACCgCA-3' and a
12-mer, 5'-gATC TgCggTgA-3'. The DNA sample was mixed with the linkers,
annealed to 50°C, and then cooled down to 10°C. Three µl of T4
DNA ligase (400 units/µl) were then added and incubated overnight at
15°C. To isolate biotinylated from nonbiotinylated DNA fragments, the
ligase-treated samples were mixed with streptavidin-coated magnetic
beads (Dynal) and gently shaken for 3 h at room temperature. After
thorough washing, the microspheres were suspended in 20 µl of 60
mM Tris-HCl, 15 mM ammonium sulfate, and 3.5
mM MgCl2 (pH 8.5) and used directly
in a 50-µl PCR reaction, using the 24-mer oligonucleotide as a
primer. Amplification was then carried out in a Perkin-Elmer Gene-Amp
PCR 9600 system, as described (16)
: 3' incubation at
72°C, followed by addition of Taq polymerase and incubation 5' at
72°C. Twenty cycles of 1 min at 95°C and 3 min at 72°C were then
performed. After a final extension for 10 min at 72°C, the products
were held at 4°C overnight. The PCR products (70% of total product)
were analyzed in a 1.5% horizontal agarose gel (3 h at 100 V) and
stained with 1 µg/ml ethidium bromide. Experiments were repeated
three to five times. Sequencing of PCR products was carried out by
excising DNA from the agarose gel (Geneclean II kit; Bio101) and
processing them on an automated sequencer.
To examine the presence of mutations in a specific genetic
region, the linker-ligation step in the above protocol can be omitted.
After isolation of ARP-biotinylated DNA molecules with
streptavidin-microspheres, PCR primers for the desired genetic region
were added, followed by amplification. To apply this approach for the
plasmid system, PCR for a 236-bp region encompassing the known TG
mutation in the plasmid was used after binding to
streptavidin-microspheres (5'-ACT CAA GGA TGC CCA GGC TG-3' forward
primer, and 5'CCT ATT GCA AGC AAG GGT TC-3' reverse primer; PCR
cycling: 95°C, 1 min and then (95°C 30 s; 68°C, 1 min) for
25 cycles using Advantage-2 polymerase (Clontech); finally, 72°C 5
min, and hold at 4°C). The presence of mutations in the isolated
p53-exon 4 from genomic DNA was similarly detected using the PCR
conditions described above. Thus, after incubation with microspheres,
PCR was applied using the same primers used for exon 4 during
amplification from genomic DNA. These experiments were repeated twice.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C:G mutant and wild-type alleles, strong
chemiluminescence signals were obtained (Fig. 2A
, Lane 3). When MutY or ARP were omitted,
almost background signals were generated (Fig. 2
A, Lanes 1
and 2). Similar to a fluoresceinated aldehyde reactive probe
on which we reported earlier (13)
, ARP also appears to
react with the MutY-generated aldehydes at positions of A/G mismatches
that are presumed to form. Strong chemiluminescence signals were also
obtained when heteroduplexes derived from cross-hybridization of
plasmid digests were MutY treated (Fig. 2A
). Therefore,
chemiluminescence can provide signals relating to the overall TG
mutations in a sample consisting of diverse DNA fragments. The
chemiluminescence signals are proportional to the amount of DNA applied
on the microplates (Fig. 2
B, inset).
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for the quantitative isolation and
PCR amplification of ARP-reacted DNA fragments, wtp53 plasmid was first
digested with Sau3A1 into 31 DNA fragments of varying sizes, ligated to
synthetic linkers, and PCR amplified. The PCR products (Fig. 3B
, Lanes 1 and 2) demonstrated similar
bands as the original digest (Fig. 3A
, Lane 2).
In the absence of primers or DNA, no PCR product was obtained (not
shown). Next, after Sau3A1 digestion the DNA was exposed for short
periods to mild acidic conditions. This exposure introduces
stoichiometric amounts of aldehyde-containing apurinic/apyrimidinic
sites in DNA (13, 15)
, which are known to couple
specifically to ARP (14, 17)
. ARP-containing fragments
were then trapped on streptavidin-coated microspheres and PCR
amplified. Fig. 3C
demonstrates a gradual increase in the
PCR products as a function of depurination. When ARP was omitted, no
PCR products were obtained (Fig. 3
C, Lane 5). In addition,
the PCR products contain bands similar to the original plasmid digest
(Fig. 3A
, Lane 2), which is consistent with the
expected (5, 12, 15)
random generation of aldehydes during
depurination.
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G
Transversion-containing DNA Fragments.G transversions in
diverse DNA fragments of unknown sequence, mutant (TG) and wild-type
(T-containing) plasmids were Sau3A1-digested and cross-hybridized to
form A/G and T/C-containing heteroduplexes. The sample was then
MutY-treated and biotinylated with ARP. Fig. 4A
depicts the PCR products obtained when heteroduplexes
(mutant + wild type) or homoduplexes (wild type + wild type) were used. A strong band is evident for the heteroduplexes,
whereas no such band is present for the homoduplexes. The PCR product
profile is strikingly different from the one in Fig. 3
, where all DNA
fragments were present in the PCR product. Examination of the p53
sequence revealed two Sau3A1 digestion sites flanking the mutated
thymidine. Therefore, including the added linkers, the
mutation-carrying fragment should be 602 bp (554 bp plasmid + 48 bp linkers), which agrees with the data in Fig. 4A
. To further determine the identity of the resulting
fragment, the DNA was recovered from the gel and sequenced in parallel
with the original wild-type and mutant sequences. Sequencing clearly
demonstrated (Fig. 4B
) the recovery of the
mutation-containing sequence out of a mixture of diverse DNA fragment
sizes, via ALBUMS.
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demonstrates that ALBUMS can isolate and amplify the
mutation-containing fragment when one mutant allele is present in up to
1000-fold excess of wild-type alleles in the original plasmid mixtures.
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G mutation in the
plasmid was amplified out of the ALBUMS-selected DNA fragments. Fig. 6A
demonstrates the isolation of a 236-bp fragment when
heteroduplex DNA populations (1:1, mutant:wild type) are formed
(Lane 1) but not when homoduplex DNA (wild-type alone) is
used (Lane 2). Next, a 399-bp region (p53 exon 4, 449 bp
including the primers) amplified from patient genomic DNA samples were
similarly examined. Fig. 6B
, Lanes 1 and
2, demonstrates the PCR product amplified from genomic DNA
from the two patient samples. These two genomic DNA samples had been
precharacterized via sequencing at the DFCI Molecular Diagnostics
Laboratory and were found to be heterozygous at codon 110 (CGTCTT,
first sample) and homozygous (second sample), respectively. Fig. 6C
demonstrates the application of ALBUMS on these two PCR
products. It can be seen that, after ALBUMS, a PCR product is obtained
for the heterozygous (Lane 1) but not for the homozygous
(Lane 2) sample.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present work demonstrates a major improvement in the central
problem of unknown mutation detection, i.e., the ability to
scan unknown and diverse DNA sequences for isolation and amplification
of a rare mismatch ("finding the needle in a haystack"). In this
novel approach, after the formation of heteroduplexes, the high
specificity of MutY for mismatched adenine is used to uniquely
introduce aldehydes at positions of mutations. Previously, radioactive,
biotinylated, or fluoresceinated hydroxylamines had been shown to
efficiently trap aldehyde-containing abasic sites (12, 17, 20, 21)
. Furthermore, our group has demonstrated that a
fluoresceinated aldehyde-reactive probe resulted in sensitive
chemiluminescent detection of MutY-generated total aldehydes in a DNA
sample (13)
. However, identification of the mutant gene is
of paramount importance; hence, methods to isolate, amplify, and
identify the mutation are urgently needed. To this end, in this work a
procedure was devised to isolate, PCR amplify, and characterize the
mutations. A biotinylated ARP (14, 17)
was used to
introduce biotin at the aldehydes formed after MutY-mediated excision
of adenine from mismatched adenine:guanine pairs. After immobilization
of biotinylated DNA on streptavidin support, the intact (complementary)
strand serves as the template for PCR. The MutY-generated aldehydes are
the only reactive sites for ARP (traces of spontaneously occurring
aldehydes are removed via hydroxylamine pretreatment). Unlike
electrophoresis or chromatography-based methods, ALBUMS relies on the
avidin-biotin interaction for mutation isolation and detection and not
on DNA fragment size discrimination. Consequently, a problem that
traditionally restricted the use of enzymes for mutation detection,
nonspecific strand cutting by the enzyme used or by contaminating
nucleases (10)
, is of no consequence to the present
approach. Fig. 5
demonstrates that a single base substitution
introduced into a 7-kb plasmid is still detectable when the
mutant:wild type ratio is 0.1%. By increasing the PCR cycles and
by overexposing the gels, the PCR product in the heteroduplexes becomes
stronger (not shown); however, nonspecific bands start appearing in the
homoduplexes, as well. Because under the conditions adopted in this
paper the 0.1% mutant dilution is only just detectable, in
practice a 0.1–1% mutant:wild type ratio should be taken as the
current limit for ALBUMS. Conventional methods rarely can detect 1
mutant allele in the presence of 10 wild-type alleles
(22)
; therefore ALBUMS extends the detection limit by a
factor 10–100. If the comparison to other methods is performed in
terms of the lowest frequency of mutated bases detectable in the
presence of nonmutated DNA, the result in Fig. 5
indicates that ALBUMS
has a detection limit of 1 mutated base in 7 Mb of DNA. Conventional
methods for detecting unknown mutations typically detect up to 1
mutated base in 3 kb of DNA (22)
, whereas DNA chips can
detect up to 1 mutated base in 15–30 kb of DNA (18)
.
Accordingly, ALBUMS improves the lowest limit for unknown mutation
scanning by two to three orders of magnitude. Base substitution
mutations that can be detected with such sensitivity are the four
transversions (TG/GT or AC/CA), because A/G mismatches will
result whenever DNA heteroduplexes are formed. In addition, MutY
recognizes A/C mismatches (23)
; therefore, (GA/AG,
CT/TC) are also potentially detectable. A number of alternative
DNA base changes should also be detectable using the present approach
in conjunction with other glycosylases, e.g., the thymidine
glycosylase from Thermophilus aquaticus (mainly G/T
mismatches but also G/G; Ref. 24
) and from HeLa cells
(mainly G/T mismatches but also A/A; Ref. 25
). The method
will currently not detect small deletions or frameshifts because these
are not recognized by glycosylases. In addition, homozygous mutations
would only be detectable upon addition of a wild-type DNA to serve as a
template for heteroduplex formation. In its present format, ALBUMS
requires a starting total (mutant plus wild type) DNA of 1 µg. This
translates to a minimum requirement for 1 ng of mutant DNA in the
sample, because 0.1% mutant DNA can be detected. ALBUMS is applicable
to any form of DNA (genomic, cDNA, or plasmid), is nonisotopic, and can
be adapted to a high throughput format.
Potential Applications.
The present development allows highly selective scanning for mutations
and/or single nucleotide polymorphisms without prejudice as to which
are the DNA sequences (genes) of interest. The method relies on the
formation of heteroduplexes from a wild-type sample (e.g.,
genomic DNA or cDNA, from noncancerous tissue) and a mutated sample
(e.g., DNA from cancerous tissue). Unlike other mutation
detection methods that use heteroduplex formation (e.g.,
denaturing gradient gel electrophoresis, chemical cleavage of mismatch,
and denaturing high-precision liquid chromatography; Ref.
10
), the heteroduplexes in ALBUMS are formed in a complex
hybridization of several DNA fragments simultaneously. Once mutated DNA
fragments are isolated and amplified via ALBUMS, identification of
the corresponding genes is feasible with established methods
(e.g., sequencing, cloning, and Southern blot). Large-scale
hybridization approaches, such as application on appropriate DNA
arrays, may also be envisioned. In this manner, point mutations in
several oncogenes, for example, could be screened in a single
experiment. Therefore, ALBUMS should enable multiplex screening of
cancer samples for mutation-containing genes or for identification of
point mutations that promote malignant transformation of cells exposed
to mutagens. GT transversions in particular are the major mutation
generated by the tobacco carcinogen benzo(a)pyrene and are
found in lung cancers of smokers (26)
. Because traces of
DNA containing GT can be detected with the present method, GT
transversion scanning in several DNA fragments simultaneously could
help identify key mutated genes leading to lung cancer or could develop
into an early detection method for such malignancies. Further
envisioned ALBUMS applications include genotyping and polymorphism
studies and the role of mutations in diseases other than cancer.
Practical applications may include screening plasmids in mutagenesis
experiments and quantification of PCR errors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by USPHS Grants
K04 CA69296 and 1R21/R33 CA83234–01 (to G. M. M.) by the NIH and by
a grant from the Starr Foundation. 
2 To whom requests for reprints should be
addressed, at Brigham/Dana Farber/Children’s Radiation Therapy,
Department of Radiation Oncology, Harvard Medical School, 75 Francis
Street, Boston, MA 02115. Phone: (617) 632-6905; Fax: (617) 632-6900;
E-mail: makri{at}jcrt.harvard.edu
3 The abbreviations used are: ARP, aldehyde
reactive probe; ALBUMS, aldehyde-linker-based ultrasensitive mismatch
scanning.
Received 11/ 5/99. Accepted 5/16/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and protein kinase C 
. Biochem. Biophys. Res. Commun, 245: 514-518, 1998.[Medline]
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