Kits and Methods for Detecting Methylated DNA

Abstract

The present invention relates to an in vitro method for detecting methylated DNA comprising (a) coating a container with a polypeptide capable of binding methylated DNA; (b) contacting said polypeptide with a sample comprising methylated and/or unmethylated DNA; and (c) detecting the binding of said polypeptide to methylated DNA. In a preferred embodiment, said method further comprises step (d) analyzing the detected methylated DNA by sequencing. Another aspect of the present invention is a kit for detecting methylated DNA according to the methods of the invention comprising (a) a polypeptide capable of binding methylated DNA; (b) a container which can be coated with said polypeptide; (c) means for coating said container; and (d) means for detecting methylated DNA.

Claims

1. An in vitro method for detecting methylated DNA comprising: (a) contacting a reagent capable of specifically binding methylated DNA with a sample comprising methylated and/or unmethylated DNA, wherein the reagent has been coated on a container; wherein the reagent comprises (i) a first polypeptide and a second polypeptide each comprising a methyl-DNA-binding domain of an MBD2 protein, a fragment of the first polypeptide and a fragment of the second polypeptide, wherein each fragment is capable of binding methylated DNA, or a polypeptide that is at least 70% homologous to the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide that is at least 70% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA; (ii) an Fc portion of an antibody; and (iii) a flexible peptide linker, wherein the first polypeptide and second polypeptide each have the methyl-DNA-binding domain of the MBD2 protein fused to the Fc portion of an antibody through the flexible peptide linker; the fragment of the first polypeptide and the fragment of the second polypeptide each fused to the Fc portion of an antibody through the flexible peptide linker; or the polypeptide that is at least 70% homologous to the first polypeptide or fragment thereof and the polypeptide that is at least 70% homologous to the second polypeptide or the fragment thereof each fused to the Fc portion of an antibody through the flexible peptide linker; and the Fc portion of the antibody fused to the first polypeptide is bonded to the Fc portion of the antibody fused to the second polypeptide; the Fc portion of the antibody fused to the fragment of the first polypeptide is bonded to the Fc portion of the antibody fused to the fragment of the second polypeptide; or the Fc portion of the antibody fused to the polypeptide that is at least 70% homologous to the first polypeptide or fragment thereof is bonded to the Fc portion of the antibody fused to the polypeptide that is at least 70% homologous to the second polypeptide or fragment thereof; and (b) detecting the binding of the reagent to methylated DNA.

2. The method of claim 1, wherein step (b) comprises restriction enzyme digestion, bisulfate sequencing, pyrosequencing, Southern Blot, or PCR.

3. The method of claim 1, wherein step (b) comprises PCR.

4. The method of claim 1, further comprising step (c) analyzing the methylated DNA.

5. The method of claim 4, wherein analyzing the methylated DNA comprises sequencing.

6. The method of claim 1, wherein the container is coated directly or indirectly with the reagent.

7. The method of claim 1, wherein the sample is from a subject.

8. The method of claim 7, wherein the subject is suspected to have hypo- and/or hypermethylated gene loci.

9. The method of claim 8, wherein the hypo- and/or hypermethylated gene loci are indicative of a cancer, tumor or metastasis.

10. The method of claim 1, wherein less than about 10 ng of methylated DNA is detected in (b).

11. The method of claim 1, wherein less than about 5 ng of methylated DNA is detected in (b).

12. The method of claim 1, wherein the reagent comprises a polypeptide or fragment thereof that is at least 80% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 80% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

13. The method of claim 1, wherein the reagent comprises a polypeptide or fragment thereof that is at least 85% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 85% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

14. The method of claim 1, wherein the reagent comprises a polypeptide or fragment thereof that is at least 90% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 90% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

15. The method of claim 1, wherein the reagent comprises a polypeptide or fragment thereof that is at least 95% homologous with the first polypeptide or fragment thereof and is capable of binding methylated DNA and a polypeptide or fragment thereof that is at least 95% homologous to the second polypeptide or the fragment thereof and is capable of binding methylated DNA.

16. The method of claim 1, wherein MBD2 is human MBD2.

17. The method of claim 1, wherein MBD2 comprises amino acids 29 to 115 of SEQ ID NO:2.

18. The method of claim 1, wherein the flexible linker comprises amino acids 116 to 129 of SEQ ID NO:2.

19. The method of claim 1, wherein the binding of the reagent to methylated DNA is dependent on the degree of methylation.

20. The method of claim 1, wherein the binding of the reagent to methylated DNA is dependent on salt concentration.

Description

[0195] The figures show:

[0196] FIG. 1: Outline of Methyl-binding (MB)-PCR. (A) The major steps of the MB-PCR procedure are illustrated. MB-PCR comprises of two separate reactions, the control-PCR reaction (P-reaction) which amplifies a candidate locus directly from a genomic template, and the methyl-CpG-binding-PCR reaction which amplifies the candidate locus from the template DNA that was previously bound by a methyl-CpG-binding polypeptide in the reaction vessel (M-reaction). In the first step, the inner walls of both reaction vessels are coated with a methyl-binding polypeptide and subsequently saturated using blocking reagents (step 2). The template DNA (genomic DNA restricted with Mse I or similar enzymes) is then added to one tube (M-reaction) and allowed to bind (step 3). In the last step, the PCR reaction mix is added directly into both tubes and 50% of template DNA previously used for the M-reaction is added to the P-reaction. After gene-specific PCR, products may be analyzed, e.g. by agarose gel electrophoresis. The term “CpG-methylation low” used in FIGS. 1 A and B comprises and particularly refers to unmethylated DNA (B) Schematic representation of the MB-PCR procedure using a recombinant methyl-binding polypeptide MBD-Fc described herein above.

[0197] FIG. 2: Detecting CpG methylation in leukaemia cell lines at three CpG-island promoters by MB-PCR. (A) Shown are: the position of CpG-dinucleotides, Mse I-restriction sites, first exons and positions of primers used to detect promoter fragments of ICSBP, ESR1, and CDKN2B (p15.sup.INK4b). (B) Representative MB-PCR results of the indicated promoters for eight different leukaemia cell lines. The P-reaction directly amplifies the genomic DNA, whereas the M-reaction only amplifies CpG-methylated DNA fragments.

[0198] FIG. 3: Methylation of the ICSBP promoter inversely correlates with ICSBP expression in leukaemia cell lines. (A) Transcription levels of ICSBP were determined by LightCycler real time PCR relative to the housekeeping gene ACTB. (B) U937 cells, treated with Decitabine (DAC) for the indicated time periods were analyzed for ICSBP expression. Results were normalized to ACTB expression. Data represent mean values±SD of two independent LightCycler analyzes.

[0199] FIG. 4: Detection of aberrant CpG methylation in AML cells. Representative MB-PCRs for ESR1, CDKN2B (p15.sup.INK4b), and ICSBP promoters of several healthy donors and AML patients.

[0200] FIG. 5: MB-PCR of the ICSBP promoter correlates with the results obtained by bisulfite sequencing. Genomic DNA derived from cell lines as well as cells of selected healthy donors and AML patients was treated with bisulfite. The indicated region of the ICSBP-gene was amplified and cloned. Several independent inserts were sequenced and results are presented schematically. Circles mark the position of CpG-dinucleotides (empty: unmethylated; filled: methylated).

[0201] FIG. 6: Sensitivity of MB-PCR. (A) MB-PCRs for ESR1, CDKN2B (p15.sup.INK4b), and ICSBP promoters from mixtures of DNA from a healthy donor (unmethylated) DNA and DNA from the cell line KG-1 (methylated in all three loci). (B) DNA from three cell lines was subjected to MB-PCR using the indicated amounts of DNA for the M-reaction (or half of the indicated amount for the P-reaction). With decreasing amounts of DNA, the number of amplification cycles during PCR (given in parenthesis) was increased. Also shown is a sample that did not include DNA (H.sub.2O).

[0202] FIG. 7: FIG. 7 shows the nucleotide sequence of plasmid pMTBip/MBD2-Fc and the protein sequence (in bold) of the MBD2-Fc bifunctional protein which is encoded by plasmid pMTBip/MBD2-Fc.

The amino acid sequence of the MBD2-Fc bifunctional protein has the following features. [0203] AA 1-28 (nt 851-934): Drosophila BiP secretion signal (leader peptide from pMT/BipN5-His vector): [0204] AA 29-115 (nt 935-1196): AA 144-230 of human MBD2 [0205] AA 116-129 (nt 1196-1237): Flexible Linker (AAADPIEGRGGGGG) [0206] AA 130-361 (1238-1933): AA99-330 of human IGHG1

[0207] FIG. 8: MB-PCR detects methylation of CpG-island promoters (A) Schematic presentation of the detected MseI-fragments (indicated as grey boxes) of ESR1, CDKN2B (p15INK4b), ICSBP, ETV3, and DDX20. The position of CpG-dinucleotides, MseI-restriction sites, transcription start site, first exon and relative position of primers are marked. (B) Shown are representative MB-PCR results of normal (unmethylated) and in vitro methylated genomic DNA for the indicated promoters. The P-reaction directly amplifies the genomic DNA, whereas the M-reaction only amplifies CpGmethylated DNA fragments.

[0208] FIG. 9: Detecting CpG methylation in leukaemia cell lines by MB-PCR. (A) Shown are representative MB-PCR results of eight different leukaemia cell lines for the indicated promoters. (B) Genomic DNA from the same cell lines was analyzed by bisulfite sequencing. The indicated region of the ICSBP gene was amplified and cloned. Several independent inserts were sequenced and results are presented schematically. Squares mark the position of CpG-dinucleotides (empty: unmethylated; filled: methylated).

[0209] FIG. 10: Detection of aberrant CpG methylation in primary AML blasts. Two for the ICSBP promoter of one representative healthy donor (N) and nine AML patients are shown together with corresponding sequencing results. (Results of bisulfite sequencing are presented as described in FIG. 9.)

[0210] FIG. 11: Expression of MBD2-F.sub.c in Drosophila Schneider-cells. Stably transfected S2 cells were seeded in Medium w/o FCS, with and w/o 500 μM CuSO.sub.4. The supernatant was collected after 4 days and precleared o/n at 4° C. using sepharose beads. 1 ml precleared supernatant was precipitated using protein A sepharose, washed, re-suspended in SDS-loading dye and subjected to SDS-PAGE. The gel was Coomassie-stained to detect precipitated protein.

[0211] FIGS. 12A and 12B: Reverse South-Western Blot. A 650 bp PCR-fragment of human ICSBP-promoter (FIG. 12A) or methylated promoter fragments (50 ng) of varying CpG-density (FIG. 12B) (number of CpG-dinucleotides/100 bp: ICSBP: 10,6; CHI3L1: 2,9; TLR2: 6,2; TLR3: 2,1) were methylated using SssI, subjected to agarose gel electrophoresis (ethidium bromide staining is shown as control) and directly blotted onto nylon membrane. Membranes were stained using MBD2-Fc, HRP-conjugated anti-human Fc and ECL as described in Example 3.

[0212] FIGS. 13A, 13B, and 13C. Salt concentration-dependent binding of CpG-methylated to MBD-Fc beads (FIG. 13A) Schematic presentation of human promoter fragments. Circles mark the position of CpG-dinucleotides (∘: unmethylated—CPM; .circle-solid. SssI methylated—CCL13, TLR2, CHI3L1). (FIG. 13B and FIG. 13C) A mixture of methylated and un-methylated fragments were bound to MBD2-Fc-sepharose (amount of MBD2-Fc/50 protein A-sepharose is given) eluted using increasing salt concentrations, purified and separated using agarose gel electrophoresis (along with ⅕ of the Input mixture). Bands were visualized with ethidium bromide and scanned using a Typhoon Imager (Pharmacia-Amersham).

[0213] FIG. 14: Enrichment of CpG-islands by MCIp. Genomic DNA (300 ng) of the indicated cell types was subjected to MCIp. The enrichment of three CpG island promoters (TLR2, p15 and ESR1) was quantified using LightCycler real-time PCR. The amount of a particular promoter fragment amplified from the MCIp-eluate is shown relative to the untreated genomic DNA-control. The p15 promoter was undetectable in THP-1 cells indicating a mutation or deletion of this gene.

[0214] FIG. 15 Sensitivity of methylated CpG-island detection by MCIp. Decreasing amounts of restricted genomic U937 DNA was subjected to MCIp. The enrichment of the two CpG island promoters (TLR2, p15) was quantified using LightCycler real-time PCR. The amount of a particular promoter fragment amplified from the MCIp-eluate is shown relative to the untreated genomic DNA-control.

[0215] FIG. 16: Principle of MB-PCR. This figure shows a schematic representation of MB-PCR.

[0216] FIG. 17: MB-PCR of TLR2, ESR1 and p15 promoters in a normal and four leukemic DNA samples. Genomic DNA (10 ng) of the indicated cell types was subjected to MB-PCR. The enrichment of three CpG island promoters (TLR2, p15 and ESR1) was detected by standard genomic PCR. The p15 promoter was undetectable in THP-1 cells indicating a mutation or deletion of this gene.

[0217] FIG. 18A-18G: MCIp detection of CpG methylation in specific CpG island promoters using real-time PCR. (FIG. 18A-C) Fractionated Methyl-CpG immunoprecipitation (MCIp) was used in combination with real-time LightCycler PCR to detect the methylation status of the indicated genes from untreated (gray bars) and SssI-methylated and MseI-restricted genomic DNA fragments (black bars). Recovered gene fragments from MCIp-eluates (NaCl-concentrations (in mM) are given in boxes above) and an equivalent amount of input-DNA were amplified by LightCycler-PCR. Values (mean±SD, n=4) of individual fractions represent the percentage of recovery and are calculated relative to the amount of PCR-product generated from the respective input-DNA (100%). Above each figure a 3 kB region of the corresponding CpG island is schematically presented. Each CpG dinucleotide is represented by a vertical line. The positions of exons are indicated as grey boxes and transcription start sites by an arrow. The white box represents a 100 bp fragment. Black boxes indicate the positions of the MseI-fragments that are detected. (FIG. 18D-G) SNRPN, TLR2, ESR1, and CDKN2B gene fragments in the high salt (1000 mM) MCIp fraction of three human myeloid leukaemia cell lines (KG-1, U937 and THP-1), as well as normal human blood monocytes (N) were analyzed by Real time PCR as above.

[0218] FIGS. 19A and 19B: Sensitivity and linearity of the MCIp approach. (FIG. 19A) Decreasing amounts of MseI-treated U937 DNA were subjected to MCIp. CDKN2B and TLR2 gene fragments were quantified as above. (FIG. 19B). MseI-treated DNA of normal human blood monocytes (N) and KG-1 cells was mixed at the indicated ratios and the mixture was subjected to MCIp and the TLR2 gene fragment was quantified using LightCycler-PCR as above

[0219] A better understanding of the present invention and of its many advantages will be seen from the following examples, offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXAMPLE 1: SINGLE-TUBE ASSAY FOR THE DETECTION OF CPG-METHYLATED DNA-FRAGMENTS USING METHYL-BINDING POLYMERASE CHAIN REACTION (MB-PCR)

[0220] This method uses an approach similar to ELISAs. A protein with high affinity for CpG-methylated DNA is coated onto the walls of a PCR-cycler compatible reaction vessel and used to selectively capture strongly methylated DNA-fragments from a genomic DNA mixture. The retention of a specific DNA-fragment (e.g. a CpG island promoter of a specific gene) can be detected in the same tube using PCR (either standard PCR or realtime PCR, single or multiplex). The degree of methylation may be estimated relative to a PCR reaction of the genomic input DNA. FIG. 1 shows a schematic representation of MB-PCR.

1. Cells, Patient Samples, DNA Preparation and Fragmentation

Cells

[0221] Peripheral blood mononuclear cells (MNC) were separated by leukapheresis of healthy donors, followed by density gradient centrifugation over Ficoll/Hypaque. Monocytes were isolated from MNC by countercurrent centrifugal elutriation in a J6ME centrifuge (Beckman, München, Germany) as described in Krause, J. Leukoc. Biol. 60 (1996), 540-545. Drosophila S2 cells were obtained from ATTC and cultured in Insect-Xpress medium (Bio Whittaker) containing 10% fetal calf serum (FCS; PAA) in an incubator at 21° C. The human myeloid leukaemia cell lines THP-1, NB-4, KG-1, K562, HL-60, and U937 were grown in RPMI 1640 medium supplemented with 10% FCS. The human myeloid leukaemia cell line Mono Mac 6 was grown RPMI 1640 medium plus 10% FCS and 1% OPI media supplement (Sigma). The human myeloid leukaemia cell line MUTZ-3 was maintained in αMEM plus 20% FCS and 10 ng/ml stem cell factor. For DNA-demethylation, U937 cells were treated with the indicated amounts of Decitabine (2-deoxy-5′-azacytidine, Sigma) for several days.

Patient Samples

[0222] Fresh peripheral blood samples and bone marrow specimens from 35 patients with newly diagnosed and untreated de novo or secondary AML were used for the study. All patients were treated according to the protocol AMLCG-2000 of the German AML Cooperative Group. The study was approved by the Institutional Ethics Committee, and written informed consent was obtained from each patient before entering the study.

DNA Preparation and Fragmentation

[0223] Genomic DNA from various cellular sources, including the cell lines described herein (e.g. KG1, U937, and THP-1), normal human monocytes (healthy donor) and frozen blast cells from a patient with AML were prepared using Blood and Cell Culture Midi Kit (Qiagen). Quality of the genomic DNA-preparation was controlled by agarose gel electrophoresis and DNA concentration was determined by UV spectrophotometry. Genomic DNA was digested with Mse I (NEB) and finally quantified using PicoGreen dsDNA Quantitation Reagent (Molecular Probes). Where indicated, DNA was in vitro methylated using Sss I methylase (NEB).

2. Generation of a Recombinant Methyl-CpG-Binding Polypeptide

[0224] A cDNA corresponding to the methyl-CpG binding domain (MBD) of human MBD2 (Genbank acc. no. NM 003927; AA 144-230) was PCR-amplified from reverse transcribed human primary macrophage total RNA using primers MBD2-Nhe_S (5′-AGA TGC TAG CAC GGA GAG CGG GAA GAG G-3′) (SEQ ID NO: 4) and MBD2-Not_AS (5′-ATC ACG CGG CCG CCA GAG GAT CGT TTC GCA GTC TC-3′) (SEQ ID NO: 5) and Herculase DNA Polymerase (Stratagene). Cycling parameters were: 95° C., 3 min denaturation; 95° C., 20 s, 65° C., 20 s, 72° C., 80 s amplification for 34 cycles; 72° C., 5 min final extension. The PCR-product was precipitated, digested with Not I/Nhe I, cloned into NotI/NheI-sites of Signal pIg plus vector (Ingenius, R&D Systems) and sequence verified resulting in pIg/MBD2-Fc (eukaryotic expression vector). To clone pMTBip/MBD2-Fc for recombinant expression in Drosophila S2 cells, the Apa I/Nhe I—fragment of pIg/MBD2-Fc containing the MBD of human MBD2 fused to the Fc-tail of human IgG1 was subcloned into Apa I/Spe I—sites of pMTBiP/V5-His B (Invitrogen).

[0225] Drosophila S2 cells were obtained from ATTC and cultured in Insect-Xpress medium (Bio Whittaker) containing 10% FCS (PAA) in an incubator at 21° C.

[0226] 4×10.sup.6 Drosophila S2 cells/60 mm cell culture dish were transfected with a mixture of 1.5 μg pMTBip/MBD2-Fc and 0.3 μg pCoHygro (Invitrogen) using Effectene transfection reagent (Qiagen) according to the manufacturers protocol. On day three, transfected cells were harvested, washed, and replated in selection medium (Insect-Xpress) containing 10% FCS and 300 μg/ml Hygromycin (BD Biosciences). Selection medium was replaced every 4-5 days for five weeks. The pool of stably transfected Drosophila S2 cells was expanded. For large scale production of the methyl-CpG binding polypeptide MBD-Fc, 1-5×10.sup.8 cells were cultured in 100-200 ml Insect-Xpress without FCS (optional: 300 μg/ml Hygromycin) in 2000 ml roller bottles for two days before the addition of 0.5 mM CuSO.sub.4. Medium was harvested every 4-7 days and cells were replated medium plus CuSO.sub.4 for further protein production. Cell culture supernatants were combined, dialysed against TBS (pH 7.4), and purified using a protein A column. The MBD-Fc containing fractions were combined and dialysed against TBS (pH 7.4). The stably transfected Drosophila S2 cells produced 3-5 mg recombinant MBD2-Fc protein per litre cell culture supernatant. The sequence and features of the MBD-Fc protein are shown in FIG. 7.

3. Preparation of MB-PCR Tubes

[0227] 50 μl of the recombinant MBD2-Fc protein comprising the methyl-CpG binding domain (MBD) of human methyl-CpG-binding domain 2 (MBD2), a flexible linker polypeptide and the Fc portion of human IgG1 (diluted at 15 μg/ml in 10 mM Tris/HCl pH 7.5) were added to each well of heat stable TopYield™ Strips (Nunc Cat. No. 248909) and incubated overnight at 4° C. Wells were washed three times with 200 μl TBS (20 mM Tris, pH 7.4 containing 170 mM NaCl) and blocked at RT for 3-4 h with 100 μl Blocking Solution (10 mM Tris, pH 7.5 containing 170 mM NaCl, 5% skim milk powder, 5 mM EDTA and 1 μg/ml of each poly d(I/C), poly d(A/T) and poly d(CG), all from Amersham). Tubes were washed three times with 200 μl TBST (TBS containing 0.05% Tween-20).

4. Binding of Methylated DNA Fragments

[0228] 50 μl Binding Buffer (20 mM Tris, pH 7.5 containing 400 mM NaCl, 2 mM MgCl.sub.2, 0.5 mM EDTA, and 0.05% Tween-20) were added to each well and 2 μl Mse I-digested DNA (5 ng/μl) was added to every second well (M-reaction). Wells were incubated on a shaker at RT for 40-50 min. Tubes were washed two times with 200 μl Binding Buffer and once with 10 mM Tris/HCl pH 8.0.

5. Detection of Methylated DNA Fragments

[0229] PCR was carried out directly in the treated and washed TopYield™ Strips. The PCR-mix (PCR Master Mix (Promega); 50 μl-reactions/well) included 10 pmol of each gene-specific primer (synthesized by Metabion). Primer sequences were P15 S (5′-GGC TCA GCT TCA TTA CCC TCC-3′) (SEQ ID NO: 6), P15 AS (5′-AAA GCC CGG AGC TAA CGA C-3′) (SEQ ID NO: 7), ESR1 S (5′-GAC TGC ACT TGC TCC CGT C-3′) (SEQ ID NO: 8), ESR1 AS (5′-AAG AGC ACA GCC CGA GGT TAG-3′) (SEQ ID NO: 9), ICSBP S (5′-CGG AAT TCC TGG GAA AGC C-3′) (SEQ ID NO: 10), ICSBP AS (5′-TTC CGA GAA ATC ACT TTC CCG-3′) (SEQ ID NO: 11), METS S (5′-AAT TGC GTC TGA AGT CTG CGG-3′), (SEQ ID NO. 12), METS AS (5′-TCC CAC ACA ACA GAG AGG CG-3′) (SEQ ID NO. 13), DP103 S (5′-GCT GTT AGT CCA GTT CCA GGT TCC-3′) (SEQ ID NO. 14), DP103 AS (5′-GTG CAA CCA CAT TTA TCT CCG G-3′) (SEQ ID NO: 15).

[0230] After adding the PCR-mix, 1 μl Mse I-digested DNA (5 ng/μl) was added to every other second well that was not previously incubated with DNA-fragments (P-reaction). PCR was performed on a MJResearch engine with the following cycling conditions: 95° C. for 3 min (denaturation), 94° C. for 20 s, 60° C. for 20 s, and 72° C. for 70 s (36 cycles) and 72° C. for 5 min (final extension). PCR-products were analyzed using 3% agarose gel electrophoresis and the ethidium bromide stained gel was scanned using a Typhoon 9200 Imager (Amersham/Pharmacia).

6. Sodium Bisulfite Sequencing

[0231] Modification of DNA with sodium bisulfite was performed as previously described. Bisulfite-treated DNA was amplified in a nested PCR reaction using the primers icsbp-out S (5′-GGG GTA GTT AGT TTT TGG TTG-3′) (SEQ ID NO: 16) and icsbp-out AS (5′-ATA AAT AAT TCC ACC CCC AC-3′) (SEQ ID NO: 17) for the first and icsbp-in S (5′-TTG TGG ATT TTG ATT AAT GGG-3′) (SEQ ID NO: 18) and icsbp-in AS (5′-CCR CCC ACT ATA CCT ACC TAC C-3′) (SEQ ID NO: 19) for the second round of amplification. PCR-products were cloned using TOPO-TA Cloning Kit (Invitrogen) and several independent clones were sequenced.

7. RNA-Preparation, Real-Time-PCR

[0232] Total RNA was isolated from different cell lines by the guanidine thiocyanate/acid phenol method (Chomczynski, Anal. Biochem. 162 (1987), 156-159. RNA (2 μg) was reverse transcribed using Superscript II MMLV-RT (Invitrogen). Real-time PCR was performed on a Lightcycler (Roche) using the Quantitect kit (Qiagen) according to the manufacturer's instructions. Primers used were: human ICSBP: sense 5′-CGT GGT GTG CAA AGG CAG-3′ (SEQ ID NO: 20), antisense 5′-CTG TTA TAG AAC TGC TGC AGC TCT C-3′ (SEQ ID NO: 21); human ACTB (β-Actin): sense 5′-TGA CGG GGT TCA CCC ACA CTG TGC CCA TCT A-3′ (SEQ ID NO: 22), antisense 5′-CTA GAA GCA TTT GTG GTG GAC GAT GGA GGG-3′ (SEQ ID NO: 23). Cycling parameters were: denaturation 95° C., 15 min, amplification 95° C., 15 s, 57° C., 20 s, 72° C., 25 s for 50 cycles. The product size was initially controlled by agarose gel electrophoresis and melting curves were analyzed to control for specificity of the PCR reactions. ICSBP data were normalized for expression of the housekeeping gene β-actin (ACTB). The relative units were calculated from a standard curve plotting 3 different concentrations of log dilutions against the PCR cycle number (CP) at which the measured fluorescence intensity reaches a fixed value. The amplification efficiency E was calculated from the slope of the standard curve by the formula: E=10.sup.−1/slope. E.sub.ICSBP was in the range of 1.87 to 1.98, E.sub.ACTB ranged from 1.76 to 1.84. For each sample, data of 3 independent analyzes were averaged.

8. Analyzing the CpG Island Methylation Status of ESR1, CDKN2B (p15.sup.INK4b) and ICSBP Promoters by MB-PCR

[0233] Several leukaemia cell lines were analyzed for their CpG island methylation status of ESR1, CDKN2B (p15.sup.INK4b), and ICSBP promoters by MB-PCR. Genomic DNA was digested with Mse I. This enzyme was chosen because it is methylation-insensitive and cuts DNA into small fragments but leaves CpG islands relatively intact. Location of the gene-specific Mse I-fragments relative to the first intron of their respective genes as well as positions of gene-specific primers used for PCR are shown in FIG. 2A. All fragments were chosen to include the putative proximal promoter regions. A total of 10 ng of restricted DNA were used for the M-reaction and 5 ng of the same digested genomic DNA were used for the P-reaction. The result of a representative MB-PCR experiment from eight different leukaemia cell lines is shown in FIG. 2B. The ESR1 promoter was amplified to varying degrees in the M-reaction of all eight samples, which is in line with previous reports demonstrating its aberrant methylation in 86% of human haematopoietic tumors. The P-reaction for the CDKN2B (p15.sup.INK4b) promoter failed completely in three cell lines (THP-1, NB-4, K562) suggesting mutations) or deletions on both alleles, which has also been demonstrated before. Two cell lines (KG-1 and MUTZ3) showed a positive M-reaction for the CDKN2B (p15.sup.INK4b) promoter, whereas three cell lines (U937, MonoMac6, HL-60) were negative. The observed results were in good concordance with previously published methylation analyzes of ESR1 and CDKN2B (p15.sup.INK4b) promoters in some of these cell lines. In some cases, P-reactions were weaker in comparison with other cell types, suggesting the loss or mutation of one allele (e.g. ESR1 in U937 cells). The ICSBP promoter was also amplified in M-reactions of six cell lines.

[0234] The degree and effect of ICSBP promoter methylation was analyzed to further validate the experimental potential of MB-PCR. Expression levels of ICSBP were analyzed in the eight leukaemia cell lines using LightCycler Real time PCR. As shown in FIG. 3A, mRNA expression levels inversely correlated with methylation degree as determined by MB-PCR. Treatment of U937 cells, which show a high degree of ICSBP promoter methylation with the demethylating agent Decitabine (5-Aza-2′Deoxycytidine) led to a marked dose- and time-dependent induction of ICSBP mRNA expression (s. FIG. 3B) indicating that the methylation-induced repression of ICSBP transcription is reversible in these cells.

[0235] To test whether MB-PCR is also able to detect the methylation of CpG island promoters in primary tumor cells, DNA was prepared from blood monocytes of healthy individuals (n=4) and blast cells of patients with AML (n=11), digested with Mse I, and subjected to MB-PCR. As shown in FIG. 4, no significant level of methylation was detected in the DNA of healthy donors, whereas most patients showed significant methylation in at least one of the three promoters analyzed.

[0236] To determine how MB-PCR results correlate with the exact pattern of CpG methylation at the ICSBP promoter, ICSBP promoter methylation was analyzed by bisulfate sequencing in selected cell lines, normal and tumor cells. The results shown in FIG. 5 indicate that the degree of promoter methylation can be predicted by MB-PCR—strong amplification signals appear to indicate a high degree, whereas weaker signals indicate a lesser degree of methylation.

[0237] Since patient samples may be contaminated with normal, potentially unmethylated cells, the effect of increasing amounts of normal DNA in a DNA sample of a tumor cell line was determined. Restricted DNA was mixed and subjected to MB-PCR. The results are shown in FIG. 6A. The signal in the M-reaction decreased in a linear fashion with increasing amounts of normal, unmethylated DNA in the sample. To test the sensitivity of the method, MB-PCR experiments using decreasing amounts of DNA were performed. As shown in FIG. 6B, comparable results were obtained using all concentrations tested (10 ng-160 pg) when analyzing the methylation status of the ICSBP locus in three different cell lines. These results indicate, that MB-PCR can detect methylated DNA-fragments in mixtures of normal cells, tumor cells, and works within the normal sensitivity range of standard genomic PCR (down to 160 pg of DNA).

9. Analyzing the CpG Island Methylation Status of ESR1, CDKN2B (p15.sup.INK4b), ICSBP, ETV3, and DDX20 Promoters by MB-PCR,

[0238] In another experiment, the MB-PCR method was explored by analyzing the degree of CpG methylation of single CpG island promoters that were previously shown to be frequently methylated in leukaemia cells, namely the human CDKN2B gene (also known as p15INK4b) and the human estrogen receptor 1 (ESR1) gene. In addition to the well established tumor markers three additional genes with CpG island promoters that could potentially act as tumor suppressor genes were selected: the human interferon consensus binding protein (ICSBP) gene, the human Ets variant 3 gene (ETV3), and the human DEAD box polypeptide 20 gene (DDX20). ICSBP, a transcription factor of the interferon (IFN) regulatory factor family (IRF), is frequently down-regulated in human myeloid leukaemia (Schmidt, Blood 91 (1991), 22-29) and ICSBP-deficient mice display hematological alterations similar to chronic myelogenous leukaemia (CML) in humans (Holtschke, Cell 87 (1996), 307-317), suggesting a tumor suppressor function for ICSBP in hemopoietic cells. In mice, the Ets repressor ETV3 (also known as METS or PE1) and its co-repressor DDX20 (also known as DP103) were shown to link terminal monocytic differentiation to cell cycle arrest (Klappacher, Cell 109 (2002), 169-180), which may also indicate a possible tumor suppressor role. As a validation of our approach, genomic DNA from normal cells was either left untreated or methylated in vitro using SssI, digested with MseI and subjected to MB-PCR. Genomic DNA was digested with MseI because this enzyme is methylation-insensitive and cuts DNA into small fragments while leaving CpG islands relatively intact (Cross, Nat. Genet. 6 (1994), 236-244). Locations of the gene-specific MseI-fragments relative to the first intron of their respective genes as well as positions of gene-specific primers used for MB-PCR are shown in FIG. 8A. All fragments include the putative proximal promoter regions. As shown in FIG. 8B, the M-reactions of all five loci were negative when normal DNA was used, indicating that these genomic regions are, as expected, free of methylation in normal blood cells. However, each locus was amplified in the corresponding M-reaction when the same DNA was in vitro methylated using SssI-methylase before it was subjected to MB-PCR. Hence, MB-PCR is able to discriminate the methylated and unmethylated state at these loci.

10. Methylation Status of Specific CpG Island Promoters in Tumor Cell Lines Analyzed by MB-PCR.

[0239] In another experiment it was tested whether MB-PCR is able to detect the methylation status of the above loci in biological samples, several leukaemia cell lines were analyzed. Routinely, a total of 10 ng of restricted DNA was used for the M-reaction and 5 ng of the same digested genomic DNA was used for the P-reaction. The result of a representative MB-PCR experiment from eight different leukaemia cell lines is shown in FIG. 9A. The ESR1 promoter was amplified to varying degrees in the M-reaction of all eight samples, which is in line with previous reports demonstrating its aberrant methylation in more than 80% of human hemopoietic tumors. The P-reaction for the CDKN2B promoter failed completely in three cell lines (THP-1, NB-4, K562) suggesting mutations or deletions on both alleles, which has been demonstrated previously in the cases of NB-4 (Chim, Ann. Hematol. 82 (2003), 738-742) and K562 (Paz, Cancer Res. 63 (2003), 1114-1121). The two cell lines KG-1 and MUTZ3 showed a positive M-reaction for the CDKN2B promoter, whereas three cell lines (U937, MonoMac6, HL-60) were negative. The observed results were in good concordance with previously published methylation analyzes of ESR1 (27) and CDKN2B promoters (Cameroon, Blood 94 (1999), 2445-2451; Chim (2003), loc. cit.; Paz (2003), loc. cit.). In some cases, P-reactions were weaker in comparison with other cell types, suggesting the loss or mutation of one allele (e.g. ESR1 in U937 cells).

[0240] Interestingly, the ICSBP promoter was also amplified in M-reactions of six cell lines, whereas no significant methylation was detected at the promoters of ETV3 and DDX20 genes.

[0241] To determine how MB-PCR results correlate with the exact pattern of CpG methylation at the ICSBP promoter in individual cell lines, the ICSBP promoter methylation was analyzed by bisulfite sequencing. The results shown in FIG. 9B indicate that the degree of promoter methylation corresponds with results obtained by MB-PCR. Strong amplification signals (comparable to the corresponding P-reaction), as seen in KG-1, U937, MUTZ-3, HL-60, and K562 cell lines, appear to indicate a high degree, whereas weaker signals (as observed for NB-4 cells) indicate a lesser degree of methylation. In the absence of DNA methylation (THP-1 and MonoMac6 cells) the MB-PCR is negative.

11. Detecting Methylation of CpG Island Promoters in Primary Tumor Cells.

[0242] DNA was prepared from blood monocytes of several healthy persons (n=4) and leukaemic blasts of patients with previously untreated AML (n=35), digested with MseI, and subjected to MB-PCR. FIG. 11 shows representative ICSBP MB-PCR and corresponding bisulfite sequencing results for 9 AML patients and 1 normal individual. In general, the intensity of the band observed in the M-reaction (as compared to the corresponding P-reaction) showed good correlation with the mean density of methylation in the sample. Out of 35 AML-patients tested, 7 patients (20%) showed positive MB-PCR results for ICSBP, 21 patients (60%) for ESR1 and 25 patients (71%) for CDKN2B (data not shown). The frequencies for ESR1 and CDKN2B methylation observed concur with those described in previous studies. ICSBP methylation apparently only affects a subgroup of patients. Twelve patients were tested for methylation of ETV3 and DDX20 genes and, as observed for the leukaemia cell lines, no significant methylation was detected in any of the samples.

EXAMPLE 2: CLONING OF PMTBIP/MBD2-FC

[0243] A cDNA corresponding to the methyl-CpG binding domain (MBD) of human MBD2 (Genbank acc. no. NM 003927; AA 144-230) was PCR-amplified from reverse transcribed human primary macrophage total RNA using primers MBD2-Nhe_S (5′-AGA TGC TAG CAC GGA GAG CGG GAA GAG G-3′) (SEQ ID NO: 4) and MBD2-Not_AS (5′-ATC ACG CGG CCG CCA GAG GAT CGT TTC GCA GTC TC-3′) (SEQ ID NO: 5) and Herculase DNA Polymerase (Stratagene). Cycling parameters were: 95° C., 3 min denaturation; 95° C., 20 s, 65° C., 20 s, 72° C., 80 s amplification for 34 cycles; 72° C., 5 min final extension. The PCR-product was precipitated, digested with Not I/Nhe I, cloned into NotI/NheI-sites of Signal pIg plus vector (Ingenius, R&D Systems), and sequence verified resulting in pIg/MBD2-Fc (eucaryotic expression vector). To clone pMTBip/MBD2-Fc for recombinant expression in Drosophila S2 cells, the Apa I/Nhe I—fragment of pIg/MBD2-Fc containing the MBD of human MBD2 fused to the Fc-tail of human IgG1 was subcloned into Apa I/Spe I—sites of pMTBiP/V5-His B (Invitrogen).

EXAMPLE 3: RECOMBINANT EXPRESSION OF AN ANTIBODY-LIKE METHYL-CPG-DNA-BINDING PROTEIN

[0244] Methylated Cytosine in single-stranded, but not double-stranded DNA molecules can be efficiently detected using 5-mC antibodies. To enable an antibody-like detection of double-stranded CpG-methylated DNA, a vector as described in Example 2 above, was constructed encoding a fusion protein comprising the methyl-CpG binding domain (MBD) of human methyl-CpG-binding domain 2 (MBD2), a flexible linker polypeptide, and the Fc portion of human IgG1. The protein was expressed under the control of a metal-inducible promoter in Drosophila S2 Schneider-cells, and collected from the supernatant via Protein A affinity chromatography. The purified protein was expressed in high amounts (4-5 mg/L cell culture supernatant) and had the expected molecular weight of appr. 40 kDa (s. FIG. 2).

[0245] Accordingly, in detail an insect cell system was chosen for recombinant expression of MBD2-Fc protein for several reason. The main reason is the absence or low abundance of CpG-methylation. Production of the protein in mammalian (especially human) cells may result in DNA contaminations (bound to the MBD2-Fc protein in the cell culture supernatant) which may complicate subsequent analysis of CpG-methylated DNA. Other reasons include the simple culture conditions and the potentially high yields of protein.

[0246] Drosophila S2 cells were obtained from ATTC and cultured in Insect-Xpress medium (Bio Whittaker) containing 10% FCS (PAA) in an incubator at 25° C.

[0247] 4×10.sup.6 Drosophila S2 cells/60 mm cell culture dish were transfected with a mixture of 1.5 μg pMTBip/MBD2-Fc and 0.3 μg pCoHygro (Invitrogen) using Effectene transfection reagent (Qiagen) according to the manufacturers protocol. On day three, transfected cells were harvested, washed, and replated in selection medium (Insect-Xpress) containing 10% FCS and 300 μg/ml Hygromycin (BD Biosciences). Selection medium was replaced every 4-5 days for five weeks. The pool of stably transfected Drosophila S2 cells was expanded and several aliquots preserved in liquid nitrogen.

[0248] For large scale production, 1-5×10.sup.8 cells were cultured in 100-200 ml Insect-Xpress without FCS (optional: 300 μg/ml Hygromycin) in 2000 ml roller bottles for two days before the addition of 0.5 mM CuSO.sub.4. Medium was harvested every 4-7 days, and cells were replated medium plus CuSO.sub.4 for further protein production. Cell culture supernatants were combined, dialysed against TBS (pH 7.4) and purified using a protein A column. The MBD-Fc containing fractions were combined and dialysed against TBS (pH 7.4). The stably transfected Drosophila S2 cells produced 3-5 mg recombinant MBD2-Fc protein per litre cell culture supernatant.

EXAMPLE 4: DETECTION OF CPG-METHYLATED DNA ON MEMBRANES (REVERSE SOUTH-WESTERN BLOT)

[0249] To test, whether MBD2-Fc was able to detect CpG-methylated DNA on membrane in a Western blot-like procedure, we blotted in vitro methylated or unmethylated PCR-fragments with different CpG density onto a Nylon-membrane using a capillary transfer system equivalent to traditional Southern blotting, however without denaturing the DNA prior to blotting. As shown in FIG. 12, using standard immunoblot conditions and MBD-Fc as an equivalent to the primary antibody, methylated DNA can be detected on Nylon membranes in a linear fashion (FIG. 12A) and depending on the CpG content (FIG. 12B). These results indicated that the MBD-Fc fusion protein is able to detect CpG-methylated DNA bound to a solid support.

EXAMPLE 5: SMALL SCALE ENRICHMENT OF CPG-METHYLATED DNA USING METHYL-CPG-IMMUNOPRECIPITATION (MCIP)

[0250] The following protocol allows a quick enrichment of CpG-methylated DNA fragments using spin columns. The DNA is bound to MBD2-Fc protein coupled to Sepharose beads via Protein A. The affinity for methylated DNA increases with the density of methylated CpG-dinucleotides and decreases with the ionic strength of the wash buffer.

5.1 Binding of the MBD2-Fc Protein to Protein a Sepharose

[0251] 8-10 μg purified MBD2-Fc protein was added to 50 μl Protein A Sepharose 4 Fast Flow beads (Amersham) in 1 ml TBS and rotated over night on a rotator at 4° C. On the next day, MBD2-Fc-beads were washed twice with buffer A (20 mM Tris-HCl pH 8.0, 2 mM MgCl.sub.2, 0.5 mM EDTA, 150 mM NaCl, 0.1% NP-40).

5.2 Restriction Digest and Quantitation of DNA

[0252] At least 1 μg genomic DNA (prepared using Qiagen columns) was digested using Mse I. Complete digest was controlled using agarose gel elecrophoresis and digested DNA was exactly quantified using PicoGreen dsDNA Quantitation Reagent (Molecular Probes).

5.3 Purification of Highly Methylated CpG-DNA

[0253] Digested DNA (300 ng) was added to the washed MBD2-Fc-beads in 1 ml buffer A and rotated for 3 h on a rotator at 4° C. Beads were transferred into SpinX-columns and spin-washed with approximately 1 ml buffer A. Beads were washed twice with 400 μl buffer B (20 mM Tris-HCl pH 8.0, 2 mM MgCl.sub.2, 0.5 mM EDTA, 450 mM NaCl, 0.1% NP-40) and twice with buffer C (20 mM Tris-HCl pH 8.0, 2 mM MgCl.sub.2, 0.5 mM EDTA, 650 mM NaCl, 0.1% NP-40). Flow through of each wash step was either discarded or collected for further analyzes. CpG-methylated DNA was eluted with 250 μl buffer D (20 mM Tris-HCl pH 8.0, 2 mM MgCl.sub.2, 0.5 mM EDTA, 1000 mM NaCl, 0.1% NP-40) into a new tube. Eluted DNA was desalted using Qiaquick Spin columns (ELUTED). In parallel, 300 ng digested DNA (INPUT) was resuspended in 250 μl buffer D and desalted using the QIAquick PCR Purification Kit (Qiagen). Both ELUTED- and INPUT-DNA was exactly quantified using the PicoGreen dsDNA Quantitation Reagent (Molecular Probes).

5.4. Alternative Approaches

[0254] DNA may be restricted using different restriction endonucleases or by sonication.

EXAMPLE 6: DETECTION AND QUANTITATION OF METHYLATED CPG-DNA FRAGMENTS GENERATED BY MCIP

[0255] To test, whether the MBD-Fc fusion protein was able to bind CpG-methylated DNA fragments in an immunoprecipitation-like approach, we first tested the binding properties of in vitro generated and differentially methylated DNA-fragments. PCR fragments of human promoters with varying CpG-density were generated using PCR (see FIG. 13) and CpG-methylated using SssI (CCL13, TLR2, CHI3L1) or left un-methylated (CPM). DNA was bound to MBD-Fc-Protein A sepharose beads in 150 mM NaCl (see. Example 5) and eluted using increasing concentrations of NaCl. Fractions were collected, spin-purified, and subjected to agarose gel electrophoresis. As shown in FIG. 13B, the affinity of a methylated fragment increased with the density of methylated CpG-dinucleotide with unmethylated DNA (CPM promoter fragment) eluting at relatively low salt concentrations and highly methylated DNA (TLR2 promoter fragment) eluting at high salt concentrations. Variation of the amount of Input-DNA did not significantly change the elution profile. However, the salt-dependent affinity of DNA was dependent on the density of the MBD-Fc fusion protein on the protein A sepharose beads. These results indicated that the MBD-Fc fusion protein is able to capture and bind CpG-methylated DNA in solution in a salt concentration- and CpG-methylation density-dependent fashion.

6.1 Quantitation on Single Gene Level Using Gene-Specific Real-Time PCR

[0256] 6.1.1 To test whether the recombinant MBD-Fc protein was able to detect the methylation density of a CpG island promoter in a complex genomic DNA mixture, genomic DNA from three leukemia cell lines and normal donor monocytes as well as blast cells from a patient with AML were restricted with Mse I and subjected to MCIp. The enrichment of three CpG island promoters (TLR2, p15 and ESR1) in the 1000 mM NaCl MCIp-fraction was detected using LightCycler-PCR. The three loci were chosen because p15 and ESR1 are known targets for methylation in leukemia and TLR2 was previously shown to be methylated in U937 cells but not in THP-1 cells. As shown in FIG. 14, none of the three loci was significantly detectable in the DNA preparation from the normal donor DNA (MO), which is consistent with a usually unmethylated state of CpG island promoters in normal cells. The enrichment of TLR2 in U937 but not in THP-1 is consistent with the previously observed methylation pattern in both cells. Bisulfite sequencing of the TLR2 promoter as described in Hähnel, J. Immunol. 168 (2002), 5629-37) demonstrated an almost complete methylation of the TLR2 promoter in KG1-cells (data not shown) which is consistent with the strong MCIp-enrichment shown in FIG. 14. The results for p15 in KG1 and U937 are consistent with published data. These data indicate that MCIp can be used to detect methylated DNA fragments of single gene fragments in genomic DNA.

[0257] Accordingly, enrichment of a specific Mse I-fragment in the MCIp eluate was detected and quantified relative to the genomic INPUT by Real-time Lightcycler-PCR. (s. FIG. 14). The enrichment may also be quantified after an unspecific DNA-amplification of both ELUTED- and INPUT-DNA (s. amplicon generation in Example 6.2.1 below, data not shown).

TABLE-US-00003 TABLE 3 Gene-specific oligonucleotide primers for CpG- island promoters Mse I fragment Antisense product Gene (bp) Sense primer primer (bp) TLR2 1358 TGTGTTTCAGGT CGAATCGAGACGC 118 GATGTGAGGTC TAGAGGC p15 699 GGCTCAGCTTCA AAAGCCCGGAGCT 87 TTACCCTCC AACGAC ESR1 1108 GACTGCACTTGC AAGAGCACAGCCC 129 TCCCGTC GAGGTTAG

[0258] In order to test whether MCIp may be used to discriminate methylated and unmethylated DNA fragments from genomic DNA, MCIp was used to enrich MseI-restricted genomic DNA of in vitro SssI-methylated and untreated normal DNA from monocytes of a healthy donor. MseI was chosen for DNA fragmentation, because it is known to preferentially cut in regions of low CpG content while leaving many CpG islands uncut (Cross, Nat. Genet. 6 (1994), 236-244).

[0259] The salt concentration-dependent enrichment of four different CpG-island promoters and a promoter with low CpG density was determined in SssI-methylated and untreated DNA relative to the input-DNA using LightCycler real-time PCR. As a positive control for DNA methylation, the SNRPN gene promoter that is subject to maternal imprinting with one of its two copies being methylated also in normal cells (Zeschnigk, Hum. Mol. Genet. 6 (1997), 387-395) was used. In normal DNA the two differentially methylated allele-fragments of SNRPN were enriched in two separate fractions (s. FIG. 18A). Only one enriched fraction was observed with SssI-methylated DNA. In the case of CDKN2B gene (also known as p15.sup.INK4b) which is known to be frequently methylated in leukaemia cells (Chim, Ann. Hematol. 82 (2003), 738-742; Dodge, Int. J. Cancer 78 (1998), 561-567; Dodge, Leuk. Res. 25 (2001), 917-925) (FIG. 18B), the fragment was detected mainly in a low salt fraction from normal DNA and in the high salt fraction from SssI-methylated DNA. Similar results were obtained for the human estrogen receptor 1 (ESR1) gene (Issa, Cancer Res. 56 (1996), 973-977) and the human Toll-like receptor 2 gene (TLR2) (data not show). As shown in FIG. 18C, the profiles of methylated and unmethylated DNA at the CHI3L1 locus were significantly different from those of the above tested CpG island promoters. Most of the untreated CHI3L1-fragment was recovered at lower NaCl concentrations, and a slight shift was observed towards higher NaCl concentrations when the DNA was SssI-methylated. Analysis of the above elution profiles suggests that: [0260] a.) A two to three hundred-fold enrichment of stronger over less methylated genomic fragments can be obtained in either low or high salt fractions; [0261] b.) Fragments with low CpG density are largely excluded from the high salt fraction. [0262] c.) The fractionated MCIp approach allows the resolution of small differences in CpG methylation density (the average difference between SssI-treated and untreated monocyte DNA is approximately six out of twelve methylated CpG residues, data not shown);

[0263] In order to test whether MCIp can detect aberrant hypermethylation in tumor samples, DNA from three leukaemia cell lines (KG1, U937, THP-1), as well as from monocytes of a healthy donor, were analyzed for SNRNP, CDKN2B, ESR1, and TLR2 promoter enrichment in the high salt fraction (s. FIG. 18D-G). The TLR2 gene promoter was enriched in KG-1 and U937 cells, but not in THP-1 or normal cells. The methylation pattern of TLR2 was confirmed by bisulfite sequencing (Haehnel, J. Immunol. 168 (2002), 5629-5637) (data not shown). Results for CDKN2B (KG-1 and U937) and ESR1 (KG-1) were also in line with previously published studies (Chim (2003); Dodge (2001); Issa (1996), all loc. cit.). None of the above three MseI fragments was significantly enriched in the DNA from normal cells. In concordance with its imprinting-related methylation status, the SNRPN gene promoter was significantly enriched in all leukaemia cell lines as well as in normal cells. These experiments established that the high salt MCIp fraction specifically enriches genomic DNA-fragments with a high degree of CpG methylation.

TABLE-US-00004 TABLE 4 Gene-specific oligonucleotide primers for real-time amplification of CpG-island promoters Gene Primer sequence (sense & antisense) SNRNP 5′-TAC ATC AGG GTG ATT GCA GTT CC-3′ 5′-TAC CGA TCA CTT CAC GTA CCT TCG-3′ TLR2 5′-TGT GTT TCA GGT GAT GTG AGG TC-3′ 5′-CGA ATC GAG ACG CTA GAG GC-3 ESR1 5′-GAC TGC ACT TGC TCC CGT C-3′ 5′-AAG AGC ACA GCC CGA GGT TAG-3′ CDKN2B 5′-GGC TCA GCT TCA TTA CCC TCC-3′ 5′-AAA GCC CGG AGC TAA CGA C-3′ CHI3L1 5′-ATC ACC CTA GTG GCT CTT CTG C-3′ 5′-CTT TTA TGG GAA CTG AGC TAT GTG TC-3′
6.1.2. In order to determine the amount of DNA required for the detection of a single gene fragment in a complex mixture of genomic DNA, decreasing amounts of DNA fragments were subjected to MCIp and subsequent LightCycler real-time PCR. As shown in FIG. 15, the methylated TLR2 promoter can be enriched and detected from as little as 1 ng genomic DNA from U937 cells. The un-methylated p15-promoter was not significantly enriched (20 ng MCIp-eluate) or not detectable (4 ng or 1 ng MCIp-eluate) in U937 cells (FIG. 15). These results indicate that MCIp is a sensitive method to detect methylated DNA-fragments in a complex genomic mixture.

[0264] In order to test the sensitivity of the approach, decreasing amounts of U937 DNA were analyzed using the MCIp approach. The enrichment of TLR2 (strong methylation) and CDKN2B gene fragments (no methylation) were determined by LightCycler real-time PCR. As shown in FIG. 19A, a significant enrichment of the TLR2 fragment was achieved using as little as 1 ng of genomic DNA fragments (equivalent to approximately 150 tumor cells) for the MCIp procedure. Samples derived from tumors may contain significant numbers of normal cells that would be expected to be unmethylated at most CpG islands. To test how linear the detection of CpG methylation is with respect to cell purity, MCIp was performed using mixtures of DNA from normal blood cells and the leukaemia cell line KG-1 showing high levels of CpG island methylation at several promoters. As shown in FIG. 19B, the TLR2 promoter fragment was only detected in samples containing KG-1 DNA and the signal gradually increased with the proportion of methylated DNA in the sample. Similar results were obtained for the ESR1 locus (data not shown). In general, most informative (with respect to effects on transcription) and clearest results (in terms of noise and background) were obtained when a target gene fragment contained only the proximal promoter within the CpG island. Also, in addition to enzyme restriction, DNA fragmentation may also be achieved by mechanical means, e.g. sonication (data not shown).

6.2 Quantitation on Genome-Wide Level Using Microarray Technology

[0265] 6.2.1 Generation of DNA-Amplicons from Genomic Mse I-Fragments Using Ligation-Mediated (Lm)-PCR

[0266] To generate a Mse I-compatible LMPCR-Linker, oligonucleotides LMPCR_S-L (5′-GCG GTG ACC CGG GAG ATC TCT TAA G-3′) and LMPCR_AS-L (5′-TAC TTA AGA GAT C-3′) were annealed as follows. Both oligos were combined at a concentration of 20 μM in nuclease-free H.sub.2O (USB), incubated at 80° C. for 10 min, and cooled down slowly to RT. The annealed Linker was stored in 50 μl-aliquots at −20° C.

[0267] LMPCR-Linker (0.5 μl/ng ELUTED- or INPUT-DNA) was ligated to the ELUTED- and in a separate reaction to an equal amount of INPUT-DNA in 60 μl reactions using 1 μl T4-Ligase (1200 u/μl, NEB) at 16° C. o/n. Linker-ligated DNA was desalted using QIAquick PCR Purification Kit (Qiagen) and eluted in 55 μl Tris-HCl pH 8.0 (5 mM).

[0268] Linker-ligated DNA (ELUTED- and INPUT separately) was PCR-amplified using LMPCR-Primer (5′-GTG ACC CGG GAG ATC TCT TAA G-3′) and Taq DNA Polymerase (Roche). The PCR mix contained 25 μl 10×PCR-buffer (Roche), 15 μl MgCl.sub.2 (25 mM, Roche), 10 μl dNTPs (10 mM each) 65 μl Betain (5M, Sigma), 2.5 μl LMPCR-Primer, 45 μl of linker-ligated DNA, 2.5 μl Taq DNA Polymerase (5 U/μl) in a total volume of 250 μl which was distributed into five PCR-tubes. Cycling parameters were: 58° C., 2 min (melting off LMPCR_AS-L), 72° C. 5 min (fill in overhangs); 95° C., 30 s, 58° C., 30 s, 72° C., 3 min amplification for 15 cycles; 72° C., 10 min final extension.

[0269] PCR-Reactions were combined and purified using QIAquick PCR Purification Kit (Qiagen). Both ELUTED- and INPUT-amplicons were exactly quantified using PicoGreen dsDNA Quantitation Reagent (Molecular Probes).

6.2.2. Analysis of MCIP-Amplicons Using CpG-Island Microarrays

[0270] MCIp-Amplicons may be analyzed using PCR (LightCycler, Standard PCR) to detect the enrichment of single gene fragments. To detect multiple gene fragments array technology may be used. The analysis of MCIp-amplicons using for example CpG island microarrays will involve the fluorescent labelling of MCIp-DNA-fragments and subsequent hybridization to microarrays using standard protocols.

EXAMPLE 7: SINGLE-TUBE ASSAY FOR THE DETECTION OF CPG-METHYLATED DNA-FRAGMENTS USING METHYL-BINDING POLYMERASE CHAIN REACTION (MB-PCR)

[0271] This method uses an approach similar to ELISAs. A protein with high affinity for CpG-methylated DNA is coated onto the walls of a PCR-cycler compatible reaction vessel and used to selectively capture strongly methylated DNA-fragments from a genomic DNA mixture. The retention of a specific DNA-fragment (e.g. a CpG island promoter of a specific gene) can be detected in the same tube using PCR (either standard PCR or realtime PCR, single or multiplex). The degree of methylation may be estimated relative to a PCR reaction of the genomic input DNA. FIG. 16 shows a schematic representation of MB-PCR.

7.1 DNA Preparation and Fragmentation

[0272] Genomic DNA from three cell lines (KG1, U937, and THP-1), normal human monocytes (healthy donor) and frozen blast cells from a patient with AML were prepared using Blood and Cell Culture Midi Kit (Qiagen). Quality of the genomic DNA-preparation was controlled by agarose gel electrophoresis and DNA concentration was determined by UV spectrophotometry. Genomic DNA was digested with Mse I (NEB) and finally quantified using PicoGreen dsDNA Quantitation Reagent (Molecular Probes).

7.2 Preparation of PCR Tubes

[0273] MBD-Fc-coated PCR tubes were prepared using heat stable TopYield™ Strips (Nunc Cat. No. 248909). 50 μl of recombinant MBD-Fc protein (diluted at 15 μg/ml in 10 mM Tris/HCl pH 7.5) were added to each well and incubated overnight at 4° C. Wells were washed three times with 200 μl TBS (20 mM Tris, pH 7.4 containing 150 mM NaCl) and blocked overnight at 4° C. with 100 μl Blocking Solution (10 mM Tris, pH 7.5 containing 150 mM NaCl, 4.5% skim milk powder, 5 mM EDTA, and 0.8 μg/ml of each poly d(I/C), poly d(A/T and poly d(CG)). Tubes were washed three times with 200 μl TBST (TBS containing 0.1% Tween-20.

7.3 Binding of Methylated DNA

[0274] 50 μl Binding Buffer (20 mM Tris, pH 7.5 containing 400 mM NaCl, 2 mM MgCl.sub.2, 0.5 mM EDTA, and 0.1% Tween-20) were added to each well, and 1 μl Mse I-digested DNA (10 ng/μl) was added to every second well (M-reaction). Wells were incubated on a shaker at 4° C. for 3 hours. Tubes were washed three times with 200 μl Binding Buffer and once with 10 mM Tris/HCl pH 7.5.

7.4 Detection of Methylated DNA Fragments

[0275] PCR was carried out directly in the TopYield™ Strips. The PCR-Mix (50 μl/well) contained a standard PCR buffer (Roche), 2.5 U FastStart Taq DNA Polymerase (Roche), 10 pmol of each gene-specific primer (synthesized by Qiagen), dNTPs (200 mM each, Amersham/Pharmacia) 1 M betaine (Sigma), primer sequences, and cycling parameters are shown in Table 5 & 6, respectively. After adding the PCR-mix, 1 μl Mse I-digested DNA (10 ng/μl) was added to every second other well, that was not previously incubated with DNA-fragments (P-reaction). PCR-products were analyzed using agarose gel electrophoresis, and the ethidium bromide stained gel was scanned using a Typhoon 9200 Imager (Amersham/Pharmacia).

TABLE-US-00005 TABLE 5 Cycling parameters (MB-PCR): 94° C. 3 min 94° C. 30 s 60° C. 30 s 37 × 72° C. 50 s 72° C. 5 min 15° C. ∞

TABLE-US-00006 TABLE 6 Gene-specific oligonucleotide primers for CpG-island promoters Mse I fragment Antisense product Gene (bp) Sense primer primer (bp) TLR2 1358 TGTGTTTCAGGT CGAATCGAGACGC 118 GATGTGAGGTC TAGAGGC p15 699 GGCTCAGCTTCA AAAGCCCGGAGCT 87 TTACCCTCC AACGAC ESR1 1108 GACTGCACTTGC AAGAGCACAGCCC 129 TCCCGTC GAGGTTAG

[0276] FIG. 17 shows the result of an MB-PCR experiment analyzing the methylation profile of three different CpG-island promoters in five cell types. The lanes marked with P represent the amplification of the genomic input DNA. With an exception of the (probably deleted or mutated) p15 gene in THP-1 cells, all promoters were amplified. Notably, none of the promoters was detected in the MB-PCR reactions from the normal DNA control, which is consistent with the fact that these promoters are not methylated in normal individuals. In the cell lines as well as in the patient sample, promoters were mostly methylated. The results correspond to the data obtained with MCIp in independent experiments.