[CANCER RESEARCH57. 1030-1034. March 15. 9971
Advances in Brief
Identification of DNA Methylation Markers for Human Breast Carcinomas Using
the Methylation-sensitive Restriction Fingerprinting Technique1
Tim Hw-Ming
Huang,2
Douglas
E. Laux,
Brian
C. Hamlin,
Phuong
Tran,
Hoa Tran,
and Dennis
B. Lubahn
Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center IT. H-M. H., B. C H., P. T., H. T.J, and Departments of Biochemistry and C'hild Health ID. E. L,
D. B. LI, School of Medicine, University of Missouri. Columbia, Missouri 65203
igenesis, abnormally hypermethylated or hypomethylated sequences
occurring in the tumor genome need to be identified.
We have developed a PCR-based method, called methylation-sensitive
In this study, we have developed a PCR-based method, called
restiiction fingerprinting (MSRF), to screen changes in DNA methylation In
MSRF,3 to identify abnormally methylated CpG sites in breast carci
breast carcInomas. Two hypennethylatios-containing fragments, HBC-1 (for
nomas. Initially, genomic DNA was digested with a 4-base restriction
“hypennethylation In breast cancer―) and HBC-2, were identified In the
endonuclease known to cut bulk DNA into small fragments but rarely
amplified breast tumor DNA relative to the amplified normal breast DNA of
to cut in the CG-rich regions. The digests were treated with a second
a patient Nucleotide sequence analysis revealed no significant matches be
restriction endonuclease, which discriminates between methylated and
tween the sequence of HBC-1 and the known sequences In the GenBank
database, whereas the sequence ofHBC-2 matched the upstream region of an unmethylated CpG sites, and then amplified by PCR with short
antisense WTJ (Wilma' tumor suppressor gene) promoter. The methylation
arbitrary primers (lO-mers) at low stringency. Amplified products or
status In the breast tumor DNA from this patient was confirmed by Southern
DNA fingerprints were resolved by high-resolution gel electrophore
Abstract
hybrklization
usIng HBC-1 and HBC-2 as probes, respectively. Further anal
sis, and aberrantly
ysis showed that HBC-1 was methylated aberrantly In 90% (17 of 19 patients)
of the primary breast carcinomas examined. This study demonstrates that
MSRF provides a useful means for screening aberrant changes In DNA
methylation
during
tumorigenesis.
The commonly
methylated
fragments
identified by MSRF could potentially supplement pathological markers cur
rently used for cancers and additionally lead to the discovery of novel
methylated
tumor suppressor
genes.
methylated
patterns
were detected
in the amplified
tumor DNA relative to the amplified normal DNA of the same patient.
Here we have successfully employed the technique to identify and
clone genomic fragments that frequently undergo methylation
changes in primary breast carcinomas.
Materials
and Methods
Tissue Samples. Tumor specimens were obtained from patients with in
Introduction
vasive
breast carcinomas
undergoing
mastectomies
or biopsies
at the Ellis
Fischel Cancer Center (Columbia, MO; this study has been approved by our
Abnormal changes in DNA methylation have been observed in
tumor cells (for a review, see Refs. 1 and 2). These changes usually
occur in CpG dinucleotides that are clustered frequently in regions of
about 1—2kb in length, called CpG islands, in or near the promoter
and first exon regions of genes (3). Hypermethylation within CpG
obtained from the same patient to serve as a control (designated as “normal―).
High molecular weight DNA was extracted using standard methods.
MSRF. Genomic DNA was digested with MseI alone or digested with
islands
mended by the supplier (New England Biolabs). The PCR reaction was
was found
to be associated
with inactivation
of tumor
sup
institutional
BstU
review
board).
The adjacent
I and MseI at 10 units per
nonneoplastic
@tgDNA
following
breast
tissue
the conditions
was also
recom
pressor genes, including the estrogen receptor gene (4) and the MDGI
(mammary-derived growth inhibitor) gene (5) in breast carcinoma, the
glutathione S-transferase gene in prostate carcinoma (6), the von
performed with the digested DNA (20—100ng) in a 20-s.d volume containing
Hippel-Lindau
(pH 8.4), 2 mM MgCl2, 5 mr@if3-mercaptoethanol,0.01% (w/v) gelatin, and
0.1% (w/v) thesiti (13). The primers were as follows: Bsl, 5'-AGCGGC
CGCG; Bs5, 5'-CTCCCACGCG; Bs7, 5'-GAGGTGCGCG; BslO, 5'-AGGG
tumor suppressor
gene in renal cell carcinoma
a pair of primers (0.4 jIM), 0.15 units of Taq DNA
(7), the
RB gene in retinoblastoma (8), and the hypermethylation in cancer 1
gene (9), the cyclin-dependent
kinase
N2/p16
gene (10, Il),
and the
E-cadherin gene (12) in several types of tumor cells. In addition,
aberrant hypermethylation may contribute to tumorigenesis through
the C-to-T mutation at the methylated CpG dinucleotides (2). Hypo
methylation was also observed in nucleotide sequences of several
proto-oncogenes. Potentially, hypomethylation is linked to aberrant
increases in the transcription of these genes. However, a direct cor
relation between hypomethylation and the up-regulation of proto
oncogene expression in tumor cells has yet to be established. As a
further step to understanding the role of DNA methylation in tumor
polymerase
(Life Tech
nologies, Inc.), 5% (v/v) DMSO, 200 p.M deoxynucleotide
triphosphates,
2
III [30 mM Tricine
@Ci[a-32P] dCTP (3000 Ci/mmol;
Amersham)
in buffer
and
GACGCG; Bsl 1, 5'-GAGAGGCGCG;
Bsl2, 5'-GCCCCCGCGA;
Bs13, 5'CGGGGCGCGA; Bs17, 5'-GQGGACGCGA;
and Bsl8, 5'-ACCCCACCCG.
Initial denaturation was for 5 mm at 94°C.DNA samples were then subjected
to 30 cycles of amplification consisting of 2 mm of denaturation at 94°C,1 mm
of annealing at 40°C,and 2 mm of extension at 72°Cin a PTC-l00 thermo
cycler (M. J. Research, Inc.). The final extension was lengthened to 10 mm.
The products (20 pA)of each amplification reaction were mixed with 5 pi of
loading dye. Four pAof each DNA sample were size fractionated on a 4.5%
nondenaturing
polyacrylamide
gel. Samples
were loaded
onto the gel at —16
V/cm to prevent diffusion from each lane into its neighboring lanes. The gel
was then run for 2 h at 60 W until the bromphenol blue dye front reached 5—10
Received I0/I 8/96; accepted 1/27/97.
cm from the bottom
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
by
NIH
grant
R29-CA-69065
(to
T.
H-M.
H.)
and
2 To
whom
requests
for reprints
should
be addressed,
at the Department
of the gel. After electrophoresis,
24—48 h with DuPont Ultra-vision
wet gels were
film.
Cloning and Sequencing. Target bands were excised from polyacrylamide
by
United States Army Medical Research Command Grant DAMD17-96-l-6055 (to D. B.
L.).
edge
wrapped with plastic film and subjected to autoradiography at —70°C
for
gels, and gel fragments were shattered by being centrifuged through a pinhole
of a microfuge tube into a second intact microfuge tube. DNA was eluted in
of Pathology
and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri, 115
Business Loop 1-70 West, Columbia, MO 65203. Phone: 573-882-1283;
5206.
Fax: 573-884-
3 The
abbreviations
used
are:
MSRF,
methylation-sensitive
restriction
fingerprinting;
HBC, hypermethylation in breast cancer; RLGS, restriction landmark genomic scanning.
1030
METHYLATION-SENSITIVE
RESTRICTION
water and reamplified by 30 cycles of PCR with appropriate primers. Unmod
ified PCR products
were ligated
directly
into the TA cloning
vector,
FINGERPRINTING
within CpG islands (once per 90 bp) but is rare in bulk DNA (one site per
5,000—10,000bp) (20); and (c) methylated CpG islands protected from
pCR II,
and transformed into Escherichia coli competent cells following the supplier's
protocol (Invitrogen). Six to eight white colonies were selected, and plasmid
DNA was isolated using minipreparation protocols (Qiagen). Double-stranded
plasmid DNA was prepared and sequenced with an automated ABI model 373
the BstU I digestion
were PCR-amplified
with short arbitrary
primers
(lO-mers) attaching to the flanking homologous sequences in the genome
in opposite directions.
Except for primer Bs18 (see “Materialsand Meth
sequencer (Applied Biosystems, Inc.). The resulting nucleotide sequence was
compared to GenBank using the BLAST program (14).
Southern Hybridization Analysis. Five @gof genomic DNA were di
ods―),these primers were designed further to contain CGCG at their 3'
ends, which would enhance the chance of amplifying regions harboring
gested with MseI alone or digested
At least four conditions related to DNA methylation are distin
guished by MSRF (Fig. 1A). In a condition showing no methylation
(condition 1), PCR products are identified only in the MseI-digested
with MseI and BstU I at high concentration
(15 units4tg) to ensure complete digestion of both tumor and normal DNAs.
The digestion
products
were separated
on a 1.5% agarose gel and transferred
to nylon membrane (Schleicher & Schuell). Filters were hybridized with a
32P-labeled HBC-l probe (230 bp) or a 32P-labeled fragment, HBC-2. 1 (119
bp; positions
21—139; see Fig. 2B). Hybridization
carried out essentially
as described
and washing
conditions
were
(15). Filters were exposed to KOdak
the BstU I sites.
tumor
and normal
DNAs
(Fig.
1 A, Lanes
2 and 4) but not in the
MseIJBstU I-digested tumor and normal DNAs (Fig. 1 A, Lanes 1 and
3). The absence of products in Lanes 1 and 3 (Fig. 1A) occurs because
BioMax film in the presence of an intensifying screen for 7 days at —70°C. the given genomic sequence, which contains an internal CGCG
site(s), is restricted with BstU I and cannot be amplified by PCR using
Results and Discussion
appropriate
primers. In condition 2, an internal CGCG site(s) for a
given
Several fingerprinting techniques based on arbitrarily primed PCR (16,
17) have previously been applied to identify genomic alterations (18) and
differentially expressed transcripts (19) in several types oftumor cells. In
the present report, we describe an improved fingerprinting
technique,
MSRF, to identify abnormally methylated CpG sites in the breast tumor
genome.
Three unique features
have been implemented
genomic
sequence
is methylated
in both
tumor
and normal
genomes and thus is protected from the BstU I digestion. Alterna
tively, the given genomic sequence does not contain an internal BstU
I recognition sequence(s). In either situation, amplified products are
present in all four lanes (Fig. 1A). An aberrant hypermethylation event
(condition 3) occurs in tumor when an amplified product is observed
to preferentially
analyze methylation changes within CpG islands: (a) the system used a
4-base cutter, MseI, which has previously been shown to restrict genomic
DNA into small fragments (once per 140 bp) but preserve the integrity of
the CpG islands (once per 1,000 bp; Ref. 20); (b) the methylation
sensitive endonuclease BstU I was used for differentiating CpG methy
lation between tumor and normal genomes. This endonuclease was
chosen because its recognition sequence (CGCG) occurs frequently
(or shows
a relative
increase
in band
intensity)
in the MseI/BstU
I-digested tumor DNA (Fig. 1A, Lane 1) but not in the double
digested normal DNA (Fig. lA, Lane 3). Amplified products are seen
in the MseI-digested
tumor
and normal
DNAs
(Fig.
1A, Lanes
2 and
4). This indicatesthat a BstU I site(s) is specifically methylatedin
tumor DNA, but not in normal
DNA. Hypomethylation
(condition
4)
is detected in tumor when a PCR product is observed in the double
B
Panel
Patient#
a
b
27
TN
47
TN
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rn
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1234
rn
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e
47
25
TN
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TN
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+-+-
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5234
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+-
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TN
+-
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Methylationconditions
in tumor DNA relative
to normalDNAGellanesThmor
Mu I/BstU
@
1
4I.
DNA
I
Mse I/BstUI
2Normal
3
Mse I
DNA
Mse I
—
1,500
methylation2.
No
Normalmethylation
sites)3.
(ornoBstUI
Hypermethylationa.
Complete—@-b.
_
Enhanced4.
, -00
Hypomethylationa.
Completeb.
‘a
Incomplete—
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——
. @2
00
Fig. 1. MSRF. A, possible outcomes of DNA methylation changes in tumor detected by MSRF. B, genomic DNAs from breast tumor (7)/normal (N) tissue pairs were treated (+)
with MseI. The digests were further treated (+) or untreated (—)with BstU I. Patient numbers are shown above lanes. The digests were separately amplified with primer pairs Bs7IBslO
(a), Bsl/Bs18 (b), Bs12/Bsl3 (c), Bs13/Bs17 (d), and BsllIBsS (e). The amplified products were size fractionated on 4.5% polyacrylamide gels. Patient numbers are shown above lanes.
Molecular weight markers (l00-bp ladder; Life Technologies, Inc.) are shown to the right. Arrowheads, hypermethylation-containing DNA fragments in tumor DNA.
1031
@
@@J:
METHYLATION-SENSITIVE
A
FINGERPRINTING
B
HBC -1
1 IAac:cIGc:cGc@IGGAGGG!rrGA
RESTRICI1ON
CTTTGCCACG
CCATTCGTTG
TGGGCTTCTA
@.rJJI
51 TGCAGT'rGTc TATAAAG'rcT GGTCAGTGATGGATGGGATTTTGGAAGATG
101 TAGTGGGTAC CAAACACAGA CCCAAGATCC ATMACAACT
GAAT'PrATI'C
151 TACAGAGCCATGGAAGGATGTGGAGGATGCGGAGGATACCCTGAC@C@
EBC-2
1 lCGGGGC@O@M
‘rc'rGATcAAA
GAACACA.AGC
CTCAGCGACCAGTAACT1'GT
@,wI
51CCCAACGCCC
c'rSGAOTACA
1.AMACTAAT
TPCM@?AACT
@C-2
.I.
101 AG'rGGAGGcA ACCCTTGGGT CTGC'N'GCGG TTCCTCCACA GGACAGTGAT
151
CCCAGATTCT CCCGAAGAAA ACCGCGG@1'T CGATT'I'CTCC AAGGC!I@
@tJJI
@rJJI
2 01
@GACAGTAACGGC'l'GGGGTA
201
@GGGTGGGG @1
T
N
I
T
II
I
I
BatUl
+-+-BstUIMieX
+
++++MaeI
1
@GGGGt2
2
N
ii
+
+
3
4
bp
@1,530
Fig. 2. A and B, upperpanels, nucleotide sequences of HBC-l and HBC-2. Both strands of the inserts were sequenced with Ti and SP6 promoter primers. Primers used in MSRF
are shown in the boxed regions. The restriction endonuclease sites for BstU I are underlined. An internal fragment, HBC-2.l(underlined), was used as a probe in Southern hybridization;
lowerpanels, methylation analysis of breast tumor-normal DNA pairs by Southern hybridization. Genomic DNA (5 @g)
was treated (+) or untreated (—)with restriction endonucleases
as indicated and subjected to Southern hybridization analysis using HBC-l and HBC-2.l, respectively, as probes. T, breast tumor, N, normal breast tissue. Molecular weight markers
(100 bp ladder; Life Technologies, Inc.) are shown to the right.
digested normal DNA (Fig. 1A, Lane 3), but not (or in a reduced
quantity) in the double-digested tumor DNA (Fig. 1A, Lane 1).
Representative results of MSRF screening using a panel of primer
We further confirmed the findings of aberrant hypermethylation in
breast tumors by Southern hybridization analysis. The HBC-l probe
detected an —-800-bpband (Fig. 2A, Lanes 2 and 4) in the MseI
pairs are shown in Fig. lB. The majority of amplified products showed
digested tumor and normal DNAs from patient 47. HBC-l also
either condition 1 (no methylation) or condition 2 (normal methylation or detected an —800-bp fragment in the MseI/BstU I-digested breast
no internal BstU I sites). Two candidate fragments containing abnormally
tumor DNA from patient 47 (Fig. 14, Lane 1) but hybridized to a band
hypermethylated BstU I sites (condition 3) in the tumor DNA were
of a smaller size (—190bp) in the digested normal DNA of the same
observed in Fig. lB. b and c at —230and —200bp, respectively (Fig. lB.
patient (Fig. 2A, Lane 3). This result indicates that the BstU I sites
arrowheads); these DNA bands were detected in the MseI/BstU 1-di
located within the —800bp MseI-digested fragment were protected
gested tumor DNA, but not in the double-digested
normal DNA. Moth
(i.e., methylated) from restriction in the tumor DNA. The —-190-bp
ylation differences between amplified tumor and normal DNAS were not
band detected corresponds to the BstU I-restricted fragment within the
noted in Fig. lB. a, d@or e. PCR products were not observed in the control
HBC-l sequence, indicating that the two BstU I sites were unmethy
reactions (no DNA template) amplified with these primer pairs at the
lated in the normal DNA. (A minor band at —-190bp was observed at
concentration specified in the protocol.
the MseIJBstU I-digested tumor DNA, which can be attributed to the
The two hypermethylation-containing
fragments (-@-23Oand —‘200
presence of contaminating normal tissue in the tumor sample.) A
bp), now designated as HBC-l and HBC-2, respectively, were cloned
similar Southern hybridization result was obtained when HBC-2.l (an
for sequencing. The fragments were found to have the expected
internal fragment of HBC-2 from the antisense WT1 promoter) was
primers at the 5' and 3' ends (Fig. 2, A and B, top). A BLAST search
used as a probe (Fig. 2B, bottom). The BstU I sites located within the
revealed no significant matches between the known sequences in the
—1000bp MseI-digested fragment were found to be methylated in the
database and the sequence of HBC-l . Subsequent observation has
digested tumor DNA (Fig. 2B, Lane 1). HBC-2 detected two frag
shown that HBC-1 contains a novel expressed sequence based on a
ments (—200and —500bp) in the digested normal DNA (Lane 3); the
Northern hybridization analysis.4
—200-bp fragment corresponds to the BstU I-digested fragment
Studies are currently underway to clone the full-length cDNA
within HBC-2, indicating that the two BstU I sites were unmethylated
related to HBC-1. HBC-2 matched the upstream region of the anti
in the normal DNA, whereas the —500-bp fragment can be attributed
sense WT1 (Wilma' tumor suppressor gene) promoter located in the
first intron on the chromosome 1lpl3 region; the 3' end of HBC-2 at to the presence of partially methylated BstU I sites in some normal
cells.
position 157—206matched the published sequence of the antisense
We extended the methylation analysis of HBC-l to an additional 18
WT1 promoter at position 1—50(GenBank accession no. S79781; Ref.
21), while the rest matched the unpublished sequence.5
4 T.
H-M.
5 K. T.
Huang,
A.
Malik,
unpublished
personal
data.
communication.
breast tumor-normal DNA pairs. Fig. 3A shows representative results of
four patients using MSRF. The HBC-l fragment was detected in the
amplified double-digested tumor DNA ofpatients 19, 25, and 65, but not
in that of patient 31. HBC-l was not noted in the double-digested normal
1032
METHYLATION-5ENSITIvE RESTRICTION FINGERPRINTING
A
Patlent#19253165TNTNTNTNrnrnrnrn@u
I+-+-+-+-+-+-+-+-A@
I++++
1234++++
1234
1234
1234
t,++++
.@++++
0
0
.@
Fig. 3. Representative results of methylation
sensitive restriction fingerprinting (MSRF) and
Southern hybridization. A, Genomic DNA from
breast tumor (7)/normal (N) tissue pairs of patients
was treated (-) or untreated (+) with restriction
endonucleases as indicated. The digests were am
.@
plified with a primer pair (Bsl and Bs18).The
amplified products were size-fractionated on 4.5%
polyacrylamide
a
gels. Patient numbers are shown at
II..
the top oflanes. Molecular weight markers (lOO-bp
ladder, Life Technologies, Inc.) are shown to the
tight.Arrowheads,the locationof a hypermethyla
tion-containing fragment HBC-l (see explanation
in Fig. lA). B, Genomic DNA (5 gig) was treated
(+) or untreated (-) with restriction endonucleases
as indicated and subjected to Southern hybridiza
tion analysis using HBC-l as a probe. T, breast
tumor, N, normal breast tissue. Patient numbers are
shown at the top of lanes. Molecular weight mark
ers (100 bp ladder; Life Technologies,
-@
—
1—
@ucHBC-1
Inc.) are
shown to the tight.
B
@uI
h@I
25
19
Patlent#
65
31
N
T
N
T
N
+
+
+
+
+
+
+
+
T
N
T
N
+
+
+
+
+
+
+
+
bp
—1,
530
h • —600
ii'
@“ —200
DNA of these patients (Residual intensifies of bands seen in the double
to determine whether hypermethylation of HBC-1 is correlated with
digested normal DNA could be due to incomplete restriction ofBstU I or
specific cinicopathological parameters of the patients with breast cancer.
low-level methylation). A parallel Southern hybridization analysis (Fig.
Hypermethylation of HBC-2 within the antisense promoter in the
intron 1 of WT1 also appeared to be common in primary breast
3B) was also conducted, showing a consistent finding of hypermethyla
carcinomas.4 We are currently extending the methylation study to the
tion detected in the tumor DNA of patients 19, 25, and 65. The digested
tumor DNA of patient 31 showed at least two smaller fragments (Fig. 3 promoter and first exon regions of WT1 . The results will be reported
B, arrowheads), —200and --‘400
bp, that can be attributed to partially
in a separate paper.
methylated or unmethylated BstU I sites within the MseI-restiicted frag
Several conditions may interfere with identification of true abnor
mal methylation sites from breast tumors. As discussed by others,
mont. This condition may reflect the heterogeneous nature ofthis primary
artifacts occur in the PCR-based methodologies such as represents
tumor or contamination of normal cells in the tumor specimen analyzed.
tional difference analysis (22) or differential display (23). As shown
The methylation data based on Southern hybridization as well as patients'
in Fig. 3A, this potential problem can be minimized by studying
clinical information are summarized in Table 1. Taken together, HBC-l
independent tumors and by scoring the same aberrant PCR products
was methylated aberrantly in 90% (17 of 19 patients) of the primary
only consistently revealed in a group of patients. Secondly, because
breast tumors examined. Currently, we are studying more tumor samples
1033
METHYLATION-SENSI11VERESTRICTION FINGERPRINTING
Acknowledgments
Table 1 Clinical information and methylation studies offemale patients with breast
tumors
The authors thank Dr. Wade V. Welshons and Christina M. Khojasteh for
status
at
Patient
critical reading of the manuscript.
tumorc364MD,IDC11Th+964MD,IDClib+1964MD,IDCIV+2542MD,IDClila+2750MD,IDCifib+2977WD,
of HBC-l in
diagnosis (yr)Histological @aClinical staging'@Methylation
no.Age
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C ÷,
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MSRF also can be applied to detect hypermethylated DNA in
tumor suppressor gene in normal and transformed human tissues correlates with gene
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possible to obtain pure tumor cells. However, the detection of hypom
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Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human
ethylation-containing fragments by MSRF can be impaired if contain
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inating normal cells are present in the tumor tissue to be tested.
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Because a small amount of genomic DNA (20—100ng) is applied in
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been developed for screening changes of CpG methylation between
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different cell types (25, 26). RLGS employed digestion of genomic
16. Williams, J. G. K., Kubelik, A. R., Livak, K. I., Rafalski, J. A., and Tingey, S. V.
DNA with a series of restriction endonucleases, one being methylation
DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.
Nucleic Acids Res., 18: 6531—6535,1990.
sensitive NotI. The digests are then subjected to two rounds of gel
17. Welsh, J., and McClelland, M. Fingerprinting genomes using PCR with arbitrary
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terns. Identification of methylation differences is based on comparison
18. Peinado, M. A., Malkhosyan, S., Velasquez, A., and Perucho, M. Isolation and
characterization of ailelic losses and gains in colorectal tumors by arbitrarily primed
of more than 2500 Not! spots between two autoradiographs represent
polymerase chain reaction. Proc. Natl. Acad. Sci. USA, 89: 10065—10069,
1992.
ing two different cell types. The procedure had the advantage of
19. Liang, P., and Pardee,A. B. Differentialdisplayofeukaryotic messengerRNA by means
surveying thousands of CpG islands in a single experiment. The
of the polymerase chain reaction. Science (Washington DC), 257: 967-971, 1992.
limitation of RLGS is that a substantial amount of genomic DNA is 20. Cross, S. H., Charlton, J. A., Nan, X., and Bird, A. P. Purification of CpG islands
using a methylated DNA binding column. Nat. Genet., 6: 236—244,1994.
required to start with, to enable the subsequent cloning of differential
21. Malik, K. T. A., Wallace, J. I., Ivins, S. M., and Brown, K. W. Identification of an
spots. For example, 500 @gof genomic DNA was used by Kawai et
antisense WT1 promoter in intron 1: implication for Wil gene regulation. Oncogene,
II: 1589—1595,1995.
a!. (25) in RLGS to clone methylation-specific DNA spots for differ
22. Lisitsyn, N., Lisitsyn, N., and Wigler, M. Cloning the differences between two
ent stages of brain development in the mouse.
complex genomes. Science (Washington DC), 259: 946—951, 1993.
MSRF, described here, provides an additional approach to study CpG
23. Watson, M. A., and Fleming, 1. P. Isolation of differentially expressed sequence tags
from human breast cancer. Cancer Res., 54: 4598—4602,1994.
methylation throughout the genome. The procedure is PCR based, only
24. Zheng, Z., Merino, M. J., Chuaqui, R., Lioua, L. A., and Emmett-Buck, M. R.
needs a small amount of genomic DNA, and, thus, is suitable for ana
Identical allelic loss on chromosome 11q13 in micmdissected in situ and invasive
lyzing clinical specimens. DNA of interest is isolated directly from gels
human breast cancer. Cancer Res., 55: 467—471, 1995.
25. Kawai, J., Hirotsune, S., Hirose, K., Fushiki, S., Watanabe, S., and Hayashizaki, Y.
and reamplified by PCR for cloning. Additionally, it should be possible
Methylation profiles of genomic DNA of mouse developmental brain detected by
to speed up methylation screening with more short primers by a high
restriction landmark genomic scanning (RLGS) method. Nucleic Acids Res., 21:
throughput protocol using a 96—wellplate format thermocycler. There
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fore, it is expected that MSRF should have a wide-ranging application in 26. Hatada, I., Sugama, S., and Mukai, T. A new imprinted gene cloned by a methylation
sensitive genome scanning method. Nucleic Acids Res., 21: 5577—5582,1993.
the study of changes in DNA methylation in cancer as well as in aging,
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cell differentiation, and development.
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