WO2006084340A1

WO2006084340A1 - Solid phase assay for methylation analysis

Info

Publication number: WO2006084340A1
Application number: PCT/AU2006/000196
Authority: WO
Inventors: Peter Laurence Molloy; Susan Margaret Mitchell
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 2005-02-14
Filing date: 2006-02-14
Publication date: 2006-08-17

Abstract

The present invention relates to a method for determining the methylation status of one or more cytosine residues in a DNA molecule, the method comprising the steps of: attaching the DNA molecule to a solid support at one or more locations along the length of the molecule such that, when attached to the solid support, the DNA molecule remains essentially free in solution; contacting the DNA molecule with at least one modifying agent to selectively modify unmethylated cytosine residues; and analysing the DNA molecule to determine the methylation status of cytosine residues in the DNA molecule,

Description

Solid Phase Assay for Methylation Analysis

Technical Field

The present invention relates to solid phase assays for the modification of nucleic acid residues, particularly the bisulphite modification of cytosine residues, in a DNA molecule. The invention further relates to methods of determining methylation status and methylation patterns in a

DNA molecule using such an assay. The invention further relates to kits for carrying out the solid phase assay and methods.

Background of the Invention

In the genomes of many organisms from bacteria through to plants and animals epigenetic modifications to nucleic acid sequences influence a range of biological processes. In eukaryotic cells the predominant epigenetic modification is DNA methylation. Specifically cytosine residues are typically enzymatically modified by methylation at the 5 position to form 5 methylcytosine. DNA methylation is associated with transcriptional silencing of genes, genomic imprinting, embryonic development and tumourigenesis, among other processes. Knowledge of the methylation status of DNA is therefore central in our understanding of a variety of biological phenomena.

Cytosine methylation is difficult to detect using conventional molecular biological techniques and is typically analysed by treatment of the DNA with bisulphite followed by selective PCR and/or sequencing. Bisulphite converts unmethylated cytosine residues to uracil; however 5 methyicytosine is much less reactive than unmethylated cytosine and remains unconverted. In subsequent amplification reactions, uracil is amplified as thymine, while the unconverted 5 methylcytosine is detectable as cytosine. Reaction conditions for treatment of DNA with sodium bisulphite and subsequent PCR amplification and sequencing were originally described by Frommer ef a/. (1992) and Clark et al. (1994), A number of variations on the reaction conditions, including entrapment of DNA in agarose beads and inclusion of denaturants in the bisulphite reaction and different methods of DNA recovery have since been described (see for example Olek et a/, 1996; Paulin et al. 1998; Boyd et al. 2004). Also a number of different methods have been applied to the analysis of bisulphite-treated DNA. These include methylation specific PCR (Herman et al. 1996), HeavyMethyl PCR (Cottrell et al. 2004) and Headloop PCR (WO 03/072810) that can be used for selective amplification of either methylated or unmethylated DNA sequences, COBRA (Xiong & Laird, 1997) and MS-SNuPE (Gonzalgo & Jones, 1997) and related techniques that examine individual sites within amplified sequences.

Treatment of DNA with bisulphite typically involves a number of distinct steps carried out in liquid phase. The DNA may first be digested or sheared to reduce size. The DNA is denatured (for example using sodium hydroxide) as cytosine residues are only reactive in single-stranded DNA and then reacted with high concentrations of sodium bisulphite (typically 2M or saturated sodium bisulphite, pH5). This reaction generates a cyclic sulphonated intermediate at each unmethylated cytosine. The solution is then de-salted to remove the bisulphite and alkali treated to convert the sulphonated derivative to uracil. The solution can then be neutralized and the DNA recovered (for example by ethanol precipitation or column purification) for subsequent analysis as described above.

Notwithstanding the widespread use of bisulphite treatment of DNA, such liquid phase reactions are typically time consuming and labour intensive. A solid phase assay would be more amenable to high throughput applications and to the use of robotics. Accordingly there is a need for the development of a suitable solid phase assays for bisulphite treatment of DNA.

One method of bisulphite treatment of DNA bound to a solid surface has recently been reported in WO 03/038121 (Berlin ef a/.). As disclosed by Berlin et a/, the DNA is attached to either an aluminium oxide surface or hydrophobic C18 surface by extended direct bonding of the phosphate backbone of the DNA molecule to the surface. Further, the assay of Berlin et al. employs conditions which vary in a number of aspects from the commonly used conditions for bisulphite treatment.

The present invention is predicated on the inventors' development of an improved solid phase assay in which the DNA to be bisulphite treated is stably attached to a solid support while remaining essentially free in solution and accessible for enzymatic and other reactions. Additionally, according to the methods of the present invention the attachment of DNA to the solid support remains stable under a range of chemical conditions.

Summary of the Invention

According to a first aspect of the present invention there is provided a method for determining the methylation status of one or more cytosine residues in a DNA molecule, the method comprising the steps of:

(a) attaching the DNA molecule to a solid support at one or more locations along the length of the molecule such that, when attached to the solid support, the DNA molecule remains essentially free in solution;

(b) contacting the DNA molecule with at least one modifying agent to selectively modify unmethylated cytosine residues; and

(c) analysing the DNA molecule to determine the methylation status of cytosine residues in the DNA molecule.

The modifying agent may be a bisulphite salt. The bisulphite salt may be sodium bisulphite or an analogue or derivative thereof. The attachment may be direct, between the DNA molecule and the surface of the solid support or indirect, via a linker(s). The linker(s) may facilitate covalent or non-covalent attachment of the DNA molecule to the solid support.

In an embodiment, one end of the DNA molecule is attached to the solid support. The 3' or 5' end of the DNA molecule may be attached to the solid support.

For non-covalent attachment, the DNA molecule may be labelled with biotin and the surface of the solid support coated with streptavidin.

For covalent attachment, the DNA molecule may be labelled with a nucleophile and the surface of the solid support activated with an appropriate leaving group. In one embodiment the nucleophile may be an amine and the leaving group may be a tosyl group.

The DNA molecule may be denatured prior to attachment to the solid support. Alternatively, the DNA molecule may be attached to the solid support in double-stranded form and subsequently denatured.

The solid support may comprise any suitable solid surface, such as a microtitre tray, magnetic bead, chip or other reaction vessel, for example.

The analysis step (c) may comprise amplification of the DNA molecule by any suitable means, such as polymerase chain reaction.

According to a second aspect of the present invention there is provided a method for determining the methylation status of one or more cytosine residues in a DNA molecule, the method comprising the steps of:

(a) attaching an end of the DNA molecule to a solid support via one or more linkers such that, when attached to the solid support, the DNA molecule remains essentially free in solution;

(b) contacting the DNA molecule with sodium bisulphite or an analogue or derivative thereof to modify unmethylated cytosine residues; and (c) analysing the DNA molecule by polymerase chain reaction to determine the methylation status of cytosine residues in the DNA molecule.

The 3' or 5' end of the DNA molecule may be attached to the solid support. The linker(s) may facilitate covalent or non-covalent attachment of the DNA molecule to the solid support. For non-covalent attachment, the DNA molecule may be labelled with biotin and the surface of the solid support coated with streptavidin. For covalent attachment, the DNA molecule may be labelled with a nucleophile and the surface of the solid support activated with an appropriate leaving group. In one embodiment the nucleophile may be an amine and the leaving group may be a tosyl group.

According to a third aspect of the present invention there is provided a method for modifying one or more cytosine residues of a DNA molecule, the method comprising the steps of: - A -

(a) attaching the DNA molecule to a solid support such that, when attached to the solid support, the DNA molecule remains essentially free in solution; and

(b) treating the DNA molecule with at least one modifying agent to selectively modify unmethylated cytosine residues. The modifying agent may be a bisulphite salt. The bisulphite salt may be sodium bisulphite or an analogue or derivative thereof.

The attachment may be direct, between the DNA molecule and the surface of the solid support or indirect, via one or more linkers. The linker(s) may facilitate covalent or non-covalent attachment of the DNA molecule to the solid support. In an embodiment, one end of the DNA molecule is attached to the solid support. The 3' or

5' end of the DNA molecule may be attached to the solid support.

According to a fourth aspect of the present invention there is provided a method for bisulphite treatment of a DNA molecule, the method comprising:

(a) attaching an end of the DNA molecule to a solid support via a linker such that, when attached to the solid support, the DNA molecule remains essentially free in solution; and

(b) treating the DNA molecule with sodium bisulphite to modify the one or more cytosine residues.

The 3' or 5' end of the DNA molecule may be attached to the solid support. The linker may facilitate covalent or non-covalent attachment of the DNA molecule to the solid support. For non-covalent attachment, the DNA molecule may be labelled with biotin and the surface of the solid support coated with streptavidin. For covalent attachment, the DNA molecule may be labelled with a nucleophile and the surface of the solid support activated with an appropriate leaving group. In one embodiment the nucleophile may be an amine and the leaving group may be a tosyl group. According to a fifth aspect of the present invention there is provided a kit for use in determining the methylation status of one or more cytosine residues in a DNA molecule, the kit comprising:

(a) a solid support and at least one linker to facilitate attachment of at least one end of the DNA molecule to the solid support; and (b) instructions for attaching the DNA molecule to the solid support and for determining the methylation status of the one or more cytosine residues.

The linker(s) may comprise streptavidin, to coat the surface of the solid support, and optionally may further comprise biotin to label the DNA molecule. The linker(s) may comprise a leaving group, to activate the surface of the solid support, and optionally may further comprise a nucleophile to label the DNA molecule.

The solid support may be supplied already coated or activated with the appropriate linker to facilitate covalent or non-covalent attachment of the DNA molecule.

According to a sixth aspect of the present invention there is provided a kit for use in determining the methylation status of one or more cytosine residues in a DNA molecule, the kit comprising:

(a) a solid support, the surface of which is coated with a linker molecule to facilitate covalent attachment of the DNA molecule to the solid support; and

(b) instructions for determining the methylation status of the one or more cytosine residues. According to a seventh aspect of the present invention there is provided a kit for use in determining the methylation status of one or more cytosine residues in a DNA molecule, the kit comprising:

(a) a solid support, the surface of which is activated with a leaving group to facilitate non- covalent attachment of the DNA molecule to the solid support; and (b) instructions for determining the methylation status of the one or more cytosine residues.

Definitions

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. As used herein the term "essentially free in solution" means that the DNA, whilst remaining securely bound to the surface of the solid support, is sufficiently free from the surface along the majority of its length so as to be accessible by other molecules, for example enzymes, enabling a variety of chemical reactions to take place. Accordingly, for present purposes, the term "essentially" includes within its scope "predominantly" in relation to the amount of the DNA molecule free in solution and thereby accessible for enzymatic and other reactions.

In the context of the present invention the terms "attach", "attached" and "attachment" are used to indicate that the DNA molecule may be bonded to the solid support via direct interaction therebetween, or indirectly via a linker.

As used herein the term "linker" means a molecular tag on the DNA molecule facilitating the attachment of the DNA molecule to a solid support. Typically the linker is the molecule or compound which physically attaches the DNA molecule to the surface of the solid support, Typically for attachment of a DNA molecule comprising such a molecular tag or "linker" the surface of the solid support is itself coated with a complementary molecular tag or "linker" or otherwise treated so as to enable the indirect attachment of the DNA molecule. Thus, by virtue of the s "linker(s)" the DNA molecule itself is indirectly attached to the solid support.

Brief Description of the Drawings

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings:

Figure 1. PCR amplification of λ phage DNA either (A) untreated, (B) treated with bisulphite o in liquid phase or (C) treated with bisulphite in solid phase bound to streptavidin coated magnetic beads. The amplification profiles show the change in the level of fluorescence (units) with increasing number of PCR cycles. The melt curves (-dF/dT v temperature) plot the negative first derivative of a fluorescence v temperature plot for the amplified products.

Figure 2. PCR amplification of AIu repeat sequences from human placental DNA either (A) s untreated, (B) treated with bisulphite in liquid phase or (C) treated with bisulphite in solid phase bound to streptavidin coated magnetic beads. The amplification profiles show the change in the level of fluorescence (units) with increasing number of PCR cycles. The melt curves (-dF/dT v temperature) plot the negative first derivative of a fluorescence v temperature plot for the amplified products. 0 Figure 3. Amplification of LINE L1 repeat sequences from human genomic DNA bound to streptavidin coated magnetic beads. Comparison of amplification profiles for (A) Sephadex G50- purified biotinylated DNA and (B) biotinylated DNA directly bound without Sephadex G50 purification. X, Msel-cut and biotinylated DNA; O, Msel-cut and biotinylated DNA bound to beads; Δ, Msel-cut and biotinylated DNA bound to beads and bisulphate treated; O, water control. 5 Figure 4. Sequential PCR amplification of bisulfite-treated biotin-streptavidin bound DNA using different primer pairs in each PCR. (A) Amplification profile and melt curve for first PCR of LINE L1 repeat sequences from bisulphite treated and untreated human lymphocyte DNA. Δ, Bead-captured DNA (890pg), not bisulphate treated; O, Bead-captured DNA (540pg), bisulphate treated; O, No DNA control. (B) Amplification profile and melt curve for second PCR of AIu repeat o sequences from bisulphite treated and untreated human lymphocyte DNA following the LINE L1 PCR in (A). Δ, Lymphocyte DNA (10ng), not bisulphite treated; O, Bead-captured DNA (270pg), bisulphite treated; O, No primer control (270pg DNA).

Figure 5. PCR amplification of SVA repeat sequences from bisulphite-treated aminated human lymphocyte DNA covalently bound to tosyl-activated magnetic beads. Amplification profile 5 (A) and melt curve (B) as for Figures 1 and 2. Best Mode of Performing the Invention

Bisulphite treatment of DNA is a widely used technique enabling the identification and analysis of methylated cytosine residues. However prior art liquid phase bisulphite assays are not amenable to high throughput analysis of methylation. A recently described solid phase assay (WO 03/038121, Berlin et al.) enables higher throughput analysis than is possible with liquid phase assays but suffers from the disadvantage that DNA is irreversibly bonded essentially along the length of the molecule (via the phosphate backbone) to the solid surface thus rendering the DNA inaccessible for a variety of reactions and unstable under a broad range of conditions, such as pH and salt concentration. As disclosed herein, embodiments of the present invention provide improved methods for bisulphite treatment of DNA compared to either the liquid phase or the above-described solid phase method. By virtue of the attachment means described herein, the DNA is attached to the solid support but remains essentially free in solution and accessible for enzymatic and other reactions. The attachment means also render the bound DNA stable under a wide range of pH and salt conditions. Further, DNA fixed to the solid support cannot re-anneal and so remains single- stranded and available for reaction. This is of particular significance for the analysis of repeated DNA sequences that re-anneal rapidly in high salt conditions.

Following attachment of the DNA to the solid support, solutions can then be exchanged readily and following bisulphite treatment the DNA is directly available for a range of analysis methods such as polymerase chain reaction (PCR) or sequencing. Using methods of the present invention successive reactions can be readily performed on the same DNA molecule attached to the solid support, by simply removing solutions from the first reaction and adding new reagents.

Accordingly, one aspect of the present invention provides a method for determining the methylation status of one or more cytosine residues in a DNA molecule, the method comprising the steps of:

The present invention also provides methods for modifying one or more residues of a DNA molecule wherein the DNA molecule is attached to a solid support at one or more locations along the length of the molecule such that, when attached to the solid support, the DNA molecule remains essentially free in solution,

Typically according to methods of the present invention the modifying agent is sodium bisulphite or an analogue or derivative thereof, such as sodium metabisulphite, for example. Alternatively, other suitable bisulphite salts may be used as the modifying agent. Bisulphite reactions can be performed according to standard techniques and variations known to those skilled in the art. Other modifying agents also capable of selectively modifying unmethylated cytosine residues may be employed.

The DNA molecule to be treated may be derived from any suitable source, for example the DNA may be genomic DNA isolated from an organism, tissue or tissue section, cell or cell line. Routine procedures for the isolation and extraction of the DNA may be used. Such techniques are well known to those skilled in the art and suitable techniques may be found, for example, in Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor, New York, 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley- Intersciences, 1992. The DNA may be sheared, digested or otherwise cleaved to reduce the size of the molecules to be attached to the solid support.

According to methods of the invention, the DNA is denatured and then coupled to the solid support, or alternatively is coupled to the solid support prior to denaturation.

The solid support may take a number of forms. For example the solid support may comprise beads, such as magnetic beads, or may comprise the surface of one or more wells of a microtitre tray. Alternatively any solid surface suitable for immobilising DNA and supporting chemical reactions involving the immobilised DNA may be used, for example a chip or any suitable reaction vessel.

The DNA may be attached to the solid support either directly or indirectly by means of the interaction between a linker molecule on the DNA and the surface of the solid support, and the bonding between the DNA and the solid support may be covalent or non-covalent. The surface of the solid support may itself be modified to facilitate attachment of the DNA molecule, for example via coating with a linker capable of interaction with the linker on the DNA molecule. As exemplified herein, the DNA molecule may be end-labelled with an appropriate linker molecule to facilitate end- attachment of the DNA molecule to the solid support, therefore ensuring that the majority of the DNA is essentially free in the surrounding solution and accessible for chemical reactions to take place whilst remaining securely attached to the solid support. Those skilled in the art will readily appreciate that the DNA molecule need not be attached to the solid support via the end of the molecule but may be attached at any one or more locations along the length of the molecule. Exemplified herein are two means of attaching a DNA molecule to a solid support for the methods of the present invention, both of which enable successful solid-phase bisulphite conversion and subsequent PCR amplification. The first exemplified attachment means is non- covalent bonding via biotin-streptavidin and the second is covalent bonding with an amine tag on the DNA allowing covalent cross-linking with a tosyl-activated solid surface. In both cases it is demonstrated that the coupling is stable to the extreme chemical conditions routinely used in the bisulphite reaction, such as high salt conditions and wide pH range. For example, in the case of biotinylated DNA it is demonstrated herein that such DNA can be bound to streptavidin-coated magnetic beads either in neutral solution or 0.3M NaOH and that binding is stable to both the alkali treatment and 2M sodium metabisulphite. Biotin may be introduced at the 3' end of double- stranded DNA through 3'-end labelling with biotin-dUTP. Alternatively, biotin can also be used to tag single-stranded DNA through 3' labelling using terminal transferase, or at the 5' end via chemical labelling, for example using biotin-maleimide after kinase treatment with thio-ATP.

It will be appreciated by those skilled in the art that a number of alternate attachment means may also be employed. For example, in the case of covalent cross-linking the DNA molecule may be tagged with any suitable nucleophile capable of reacting with a carbon atom bearing a leaving group on the solid surface. For example, the nucleophile may be a mercapto group and the leaving group may be a mesylate or halide. Other suitable nucleophiles and leaving groups are known to those skilled in the art. Similarly, alternatives to biotin-streptavidin linkage may be used to non- covalently attach the DNA to the solid surface, for example digoxygenin-labeled DNA attached via an anti-digoxygenin antibody on the solid support.

Further, it will be understood by those skilled in the art that 'coating' or 'activating' the surface of the solid support as described above need not involve completely or even substantially coating or activating the entire surface. Rather the surface of the solid support need only be coated or activated to a sufficient extent to enable attachment of a sufficient amount of DNA to be treated.

Subsequent to treatment, for example with sodium bisulphite, the immobilised DNA may be subjected to one or more reactions, for example to analyse the methylation status of cytosine residues in the molecule. As exemplified herein, bisulphite-treated DNA may be amplified and the extent of methylation detected by fluorescence-based real time quantitative PCR. Further, different DNA sequences can be sequentially amplified from the same sample of treated immobilised DNA.

A number of other PCR-based methylation detection methods well known to those skilled in the art may equally be employed. Indeed the analysis need not be by way of PCR amplification and it will be clear to the skilled addressee that the scope of the present invention is not limited by the choice of analysis method used. The present invention also provides kits for carrying out the methods of the invention. In one embodiment, a kit of the present invention comprises (a) a solid support and a linker to facilitate attachment of at least one end of the DNA molecule to the solid support; and (b) instructions for attaching the DNA molecule to the solid support and for determining the methylation status of one or more cytosine residues. The solid support may be supplied already coated or activated with the appropriate linker to facilitate covalent or non-covalent attachment of the DNA molecule. A kit of the invention may further comprise a complementary linker to label a DNA molecule thereby facilitating the indirect covalent or non-covalent attachment of the DNA molecule to the solid support. A kit of the invention may additionally include other components for performing methylation studies including, for example, a modifying agent, DNA sample preparation reagents, appropriate buffers, salts and/or enzymes. The kit may further include the necessary reagents for carrying out analysis of the treated DNA, such as an appropriate reagents for polymerase chain reaction and/or reagents for analysis of amplified DNA.

The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

PCR primers and PCR amplification conditions used in the following Examples are listed in Tables 1 and 2 respectively.

Table 1 PCR Primers

I = inosine Table 2 PCR Reaction Conditions¹

1 All reactions were perfomed in 25 μl Platinum Taq Buffer containing 1.5 mM MgCk, 200 μM dNTPs, 1 μl of a 1/1000 dilution of SybrGreen stock (Molecular Probes) and 0.1 μl Platinum Taq (Invitrogen/Life Technologies).

Example 1 : Liquid phase and solid phase bisulphite treatment of λ DNA

Amplification of λ phage sequences from DNA that had been bisulphite-treated in liquid phase was compared with that from DNA coupled via biotin-streptavidin to magnetic beads.

A mixture of 5 μg human placental DNA and 100ng λ phage DNA was digested with the restriction enzyme Msel. λ phage DNA had been spiked into human placental DNA to provide a template to monitor the efficiency of bisulphite conversion. PCR primers to λ phage DNA were designed to sites lacking cytosine bases such that they would amplify DNA independently of whether DNA had been treated with bisulphite (see Table 1).

One tenth of the mix was end-filled in a 20μl reaction that contained biotin-16-dUTP at

100μM, 10μM dATP and 10 units of DNA polymerase 1, Klenow fragment. (The inventors have subsequently determined that end-filling can be more efficiently achieved using Sequenase enzyme.) Incubation was at room temperature for 20 min. Biotinylated DNA was purified using a

1mL G-50 Sepharose column, ethanol precipitated and resuspended in 20μL H₂O.

Aliquots of DNA containing 62.5 ng of placental DNA and 1.25 ng of λ phage DNA were then bisulphite-treated in liquid or solid phase as follows: (i) Liquid phase bisulphite reaction

To 18uL of DNA (62.5ng placental DNA -1 ,25ng λ phage DNA) was added 2 μl of 3M NaOH and the solution incubated for 15 min at 37⁰C. 208μL saturated sodium metabisulphite pH5.0 and 12μL 1OmM Quinol were added, the solution was overlayed with mineral oil and incubated for 8h at 55⁰C. After removing mineral oil, 1mL Wizard Resin (Promega) was added and the suspension applied to a Wizard minicolumn. The column was then washed with 80% isospropanol and DNA eluted with 50μL H2O. 5.5μL of 3M NaOH was added and the solution incubated for 15 min at 37⁰C. The solution was then neutralized by addition of NH₄OAc to 3M₁ DNA ethanol precipitated and resuspended in 20μL TE (10 mM Tris-HCL pH8, 0.1 mM EDTA). (ii) DNA binding and solid phase bisulphite reaction

5μl streptavidin beads (Dynal M-280) were washed in 100μL 2M NaCI, 0.3M NaOH, 1OmM

5 Tris, 1mM EDTA and resuspended in 20μL of the same buffer. 20μi_ of DNA (62.5ng placental

DNA-1 ,25ng λ DNA) was incubated in 0.3M NaOH for 5 min at 37⁰C. Streptavidin beads and denatured DNA were combined and incubated with shaking for 20 min at room temperature. Beads were washed twice with 100μl_ 0.3M NaOH and resuspended in 20μL of 0.3M NaOH. 208μL of saturated sodium metabisulfphite pH 5.0 and 12μL 1OmM Quinol were added, overlayed with o mineral oil and incubated for 5h at 55⁰C. After removing mineral oil, beads were rinsed with 100 μl of TE. Beads were then resuspended in 50μL 0.3M NaOH, and incubated for 15 min at 37⁰C.

Beads were then rinsed twice with 100 μl of TE and resuspended in 20 μL of same.

All solution exchanges described above were done by placing the tube containing the DNA and Dynal beads against magnet for 30 seconds, removing first solution, replacing with a new s solution and removing from the magnet. (iii) PCR amplifications

Liquid or solid-phase bisulphite-treated DNA or untreated DNA was PCR amplified in 25 μl reactions containing 62.5ng placental DNA and 1.25ng λ phage DNA. Reaction and cycling conditions are shown in Table 2 above. o For bisulphite-treated DNA from the liquid phase reaction PCR was done for 30 cycles. For the bead-bound DNA PCR was done for 5 cycles and then 2 μl of the reaction transferred to a fresh 25 μl reaction for a further 30 cycles because the presence of magnetic beads interfered with the read-out from the PCR.

SybrGreen fluorescence was monitored and melting curves determined on a Corbett 5 Research RotorGene PCR machine. The data shown in Figure 1 show that λ phage DNA modified by bisulphite in either liquid (Figure 1B) or solid phase (Figure 1C) amplified with similar efficiency. Melting profile analysis of PCR products showed that both had similar melting profiles with melting temperatures (T_m) significantly below that of unmodified λ phage DNA. This indicates efficient conversion by bisulphite of unmethylated cytosines in bead-bound DNA. o Endogenous AIu repeat sequences from human placental DNA were also amplified from the same DNA mix using primers and conditions shown in Table 2 (AIuI). The solid phase PCR was run for 15 cycles and then 1 μL taken into a fresh 25 μL reaction as above for 30 further cycles. Again a similar efficiency of amplification is evident for both liquid and solid-phase bisulphite- reacted DNA (Figure 2). Relative to the untreated control DNA (Figure 2A) both liquid phase (Figure 2B) and solid phase (Figure 2B) bisulphite-treated samples show an equivalent lowering of Tm indicative of efficient bisulphite conversion.

Example 2: Shortened protocol omitting purification of biotinylated DNA

A simplified protocol based on the above-described method of Example 1 was developed in which the Sephadex G50 purification of biotinylated DNA was omitted prior to binding to strepatavidin beads. For this protocol the concentration of biotin-dUTP is reduced to 20μM to minimise competition for binding from unincorporated biotin. Sequenase Version 2.0 DNA

Polymerase is also used as the inventors have found it provides more efficient end-filling for present purposes than the Klenow fragment of DNA Polymerase I. Msel-digested DNA was end- filled with biotin dUTP and bound to beads as follows:

250ng of Msel digested human genomic DNA was incubated at 37⁰C for15 min in a 20μL reaction in Sequenase Reaction buffer (Amersham) containing 20μM Biotin-16-dUTP (Roche) and

50μM dATP (other dNTPs included depending on digest) and 1μL (13U) Sequenase Version 2.0

DNA Polymerase (Amersham). 8μL (100ng) of end-filled DNA was denatured in 0.3M NaOH in 30μL total volume.

The denatured DNA was combined with 10μL streptavidin coated Dynabeads M-280 that had been washed in Binding buffer (2M NaCI, 0.3M NaOH₁ 1OmM Tris pH8.5,1mM EDTA) and resuspended in 30μL of Binding buffer containing 1μg of poly dl-dC (Amersham).

After incubation with shaking for 20 min at room temperature beads were washed twice with 400μL 1OmM Tris,0.1mM EDTA pH 8.0 (TE) containing 2μg tRNA and resuspended in 10μL TE (10ng/μL).

Bead-bound DNA was then bisulphite treated as described in Example 1.

The amplification of bead-bound DNA with or without treatment with sodium bisulpite was compared for biotin-labelled DNA that was bound to streptavidin beads directly after endfilling or had been purified using Sephadex G50 as in Example 1. Amplifications were done using the LINE L1 primers and reaction conditions as in Table 2. Aliquots representing 10 ng of starting DNA were amplified for an initial 5 cycles, diluted 1:5 with water and then 2 μl_ of each reaction was transferred to a fresh 25 μL reaction and amplification monitored using SybrGreen (Figure 3).

As shown in Figure 3, the amplification profiles obtained were very similar for DNA that had been purified prior to binding to the beads (Figure 3A) or was bound directly without prior purification (Figure 3B). In both cases there was a delay of about 3 cycles relative to the unbound starting DNA, reflecting a combination of incomplete binding and the inhibitory effect of the magnetic beads on the PCR. After bisulphite treatment, both purified and unpurified bound DNAs also amplified with similar kinetics, though more slowly than the non-treated DNA. This data shows th at it is possible to prepare DNA₁ end-label with biotin, bind to magnetic streptavidin-coated beads and efficiently treat with bisulphite without need for any intermediate purification steps.

Example 3: Sequential PCR amplifications using Biotin-Streptavidin Bound DNA The inventors next evaluated whether biotinylated DNA bound to streptavidin magnetic

5 beads could be used for further rounds of amplification after removing the initial PCR solution. The ability to do so would clearly indicate that would require that the binding of the DNA to the beads was not only stable through alkali and bisulphite treatments but also through the heating cycles of the PCR. Human lymphocyte DNA (Roche) was cut with Msel, biotinylated and bound to magnetic beads as in Example 1. PCR of DNA with and without bisulphite treatment was initially done using o primers to LINE repeat DNA sequences that amplify DNA independently of whether cytosines have been reacted with sodium bisulphite (ie primer target sites chosen to avoid presence of cytosines). After 10 cycles of amplification the reaction mix was removed from the beads and 2μl taken for further rounds of amplification of LINE1 sequences (reactions from cycle 10 shown in Figure 3A). The bisulphite-reacted beads were washed twice with 50μl PCR buffer without primers and s resuspended in a 25μl reaction mix for amplification of AIu sequences (Table 2, Alu(2)). A control reaction containing 10ng of unreacted lymphocyte DNA was also carried out. After 10 cycles of amplification 2μl of the reactions was transferred to fresh 25μl reactions and PCR continued for 37 cycles. A control without primers was included to confirm that amplification was not due to carryover primers from the first PCR. AIu primers FP006 and FP008 are specific for bisulphite- o converted DNA and biased toward amplification of methylated sequences expected to predominate in white blood cell DNA. As shown in Figure 4, for both the LINE amplification and the AIu amplification, the melting profiles of PCR products indicated bisulphite conversion of the DNA. Amplification of the AIu sequences demonstrates that sequential PCR amplifications using different primers can be done on DNA that has been bisulphite-treated while coupled to magnetic beads. 5 Example 4: Bisulphite treatment and amplification of covalently-coupled aminated DNA

Examples 1 to 3 demonstrate that biotin-streptavidin non-covalent linkage can be used to attach DNA to a solid support for subsequent bisulphite treatment. To demonstrate that other linker molecules can also be utilised, human lymphocyte DNA (Roche) was digested with Msel and end- filled using 5-aminoallyl-2'-deoxyuridine-5'-triphosphate (Tri-Link Biotechnologies). Reaction o conditions were as follows:

500ng of Msel-digested DNA was included in a 20μl reaction in Sequenase buffer containing 100μM 5'- aminoallyl-2'-deoxyuridine-5'-triphosphate and 13U Sequenase Version 2.0 DNA polymerase. Incubation was for 15 min at 37⁰C followed by addition of dATP to 20μM and a further 5 min incubation. Aminated DNA was purified using a 1 mL G-50 Sepharose column, ethanol 5 precipitated and resuspended in 20μL water. 10-20μL Tosylactivated beads (Dynal M-280) were washed twice in 100μL 0.2M borate buffer pH 8.3 and resuspended in 100μL of the same buffer. 50μL containing 500ng aminated DNA was incubated in 0.3M NaOH for 5 min at 37°C. Tosyl-activated beads and denatured DNA were combined and incubated with shaking for 2 h at 37°C (alternate - overnight at 4°C). Beads were washed twice with 100μL TE and resuspended in 20μL of the same. An aliquot containing 225ng of aminated DNA (9 μL) was then bisulphite-treated in solid phase as in Example 1 and resuspended in 10 μL of TE. All solution exchanges were done by placing the tube against the magnet for 30 seconds, removing the first solution, replacing with a new solution and removing from magnet.

2μL (~45ng) of the bisulphite-treated, aminated DNA covalently coupled to the tosyl-activated magnetic beads was then amplified using primers to the SVA repeat sequence family using conditions shown in Table 2. Primers F2T and R2T (Table 1) were designed such that they would amplify DNA independently of whether DNA had been treated with bisulphite.

SybrGreen fluorescence was monitored and melting curves determined on a Corbett research RotorGene PCR machine. Amplification of SVA sequences from bound, aminated DNA is shown in Figure 5; the melting curve of the product is as expected for the SVA PCR product thereby demonstrating that the covalent linkage of aminated DNA to a tosyl-activated solid support can be used to achieve efficient bisulphite conversion of unmethylated cytosines in a solid phase assay.

References

Boyd VL, Zon G. (2004) Bisulfite conversion of genomic DNA for methylation analysis: protocol simplification with higher recovery applicable to limited samples and increased throughput. Anal Biochem. 326:278-280. Clark SJ, Harrison J, Paul CL, Frommer M (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22:2990-2997.

Cottrell SE, Distler J, Goodman NS, Mooney SH, Kluth A, Olek A, Schwope I, Tetzner R, Ziebarth H, Berlin K. (2004) A real-time PCR assay for DNA-methylation using methylation-specific blockers. Nucleic Acids Res. 32:e10. Frommer, M., McDonald, L.E., Millar, D.S., Collis, CM., Watt, F., Grigg, G.W., Molloy, P.L and Paul, CL. (1992). A genomic sequencing protocol which yields a positive display of 5-methyi cytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89: 1827-1831.

Gonzalgo ML, Jones PA. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25:2529-2531.

Herman JG₁ Graff JR, Myohanen S, Nelkin BD, Baylin SB. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 93:9821-9826.

Olek A, Oswald J, Walter J (1996) A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res.24:5064-5066 Paulin R, Grigg GW, Davey MW, Piper AA (1998) Urea improves efficiency of bisulphite- mediated sequencing of 5'-methylcytosine in genomic DNA. Nucleic Acids Res.26:5009-5010.

Xiong Z, Laird PW. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25:2532-2534.

Claims

1. A method for determining the methylation status of one or more cytosine residues in a DNA molecule, the method comprising the steps of:

(a) attaching the DNA molecule to a solid support at one or more locations along the length of the 5 molecule such that, when attached to the solid support, the DNA molecule remains essentially free in solution;

(c) analysing the DNA molecule to determine the methylation status of cytosine residues in the o DNA molecule.

2. The method of claim 1 wherein the modifying agent is a bisulphite salt.

3. The method of claim 2 wherein the bisulphite salt is sodium bisulphite or an analogue or derivative thereof.

4. The method of any one of claims 1 to 3 wherein the attachment is direct, between the DNA s molecule and the surface of the solid support.

5. The method of any one of claims 1 to 3 wherein the attachment is indirect, via one or more linkers.

6. The method of claim 5 wherein the linker(s) facilitates covalent attachment of the DNA molecule to the solid support. o

7. The method of claim 5 wherein the linker(s) facilitates non-covalent attachment of the DNA molecule to the solid support,

8. The method of claim 6 wherein the DNA molecule is labelled with a nucleophile and the surface of the solid support activated with an appropriate leaving group.

9. The method of claim 8 wherein the nucleophile is an amine and the leaving group is a tosyl ₅ group.

10. The method of claim 7 wherein DNA molecule is labelled with biotin and the surface of the solid support coated with streptavidin.

11. The method of any one of claims 1 to 10 wherein one end of the DNA molecule is attached to the solid support. o

12. The method of claim 11 wherein the 3' end of the DNA molecule is attached to the solid support.

13. The method of claim 11 wherein the 5' end of the DNA molecule is attached to the solid support.

14. The method of any one of claims 1 to 13 wherein the DNA molecule is denatured prior to ₅ attachment to the solid support.

15. The method of any one of claims 1 to 13 wherein the DNA molecule is attached to the solid support in double-stranded form and subsequently denatured.

16. The method of any one of claims 1 to 15 wherein the solid support comprises a microtitre tray, magnetic bead, chip or other reaction vessel.

5 17. The method of any one of claims 1 to 16 wherein the analysis step (c) comprises amplification of the DNA molecule by polymerase chain reaction.

18. A method for determining the methylation status of one or more cytosine residues in a DNA molecule, the method comprising the steps of:

(a) attaching an end of the DNA molecule to a solid support via one or more linkers such that, when o attached to the solid support, the DNA molecule remains essentially free in solution;

(b) contacting the DNA molecule with sodium bisulphite or an analogue or derivative thereof to modify unmethylated cytosine residues; and

(c) analysing the DNA molecule by polymerase chain reaction to determine the methylation status of cytosine residues in the DNA molecule. s

19. The method of claim 18 wherein the 3' end of the DNA molecule is attached to the solid support.

20. The method of claim 18 wherein the 5' end of the DNA molecule is attached to the solid support.

21. A method for modifying one or more cytosine residues of a DNA molecule, the method o comprising the steps of:

(b) treating the DNA molecule with at least one modifying agent to selectively modify unmethylated cytosine residues. s

22. A method for bisulphite treatment of a DNA molecule, the method comprising:

(a) attaching an end of the DNA molecule to a solid support via one or more linkers such that, when attached to the solid support, the DNA molecule remains essentially free in solution; and

23. A kit for use in determining the methylation status of one or more cytosine residues in a DNA 0 molecule, the kit comprising:

(a) a solid support and at least one linker to facilitate attachment of at least one end of the DNA molecule to the solid support; and

(b) instructions for attaching the DNA molecule to the solid support and for determining the methylation status of the one or more cytosine residues.

24. The kit of claim 23 wherein the linker(s) comprises streptavidin, to coat the surface of the solid support, and optionally biotin to label the DNA molecule.

25. The kit of claim 23 wherein the linker(s) comprises a leaving group, to activate the surface of the solid support, and optionally a nucleophile to label the DNA molecule. s 26. A kit for use in determining the methylation status of one or more cytosine residues in a DNA molecule, the kit comprising:

(b) instructions for determining the methylation status of the one or more cytosine residues. o 27. A kit for use in determining the methylation status of one or more cytosine residues in a DNA molecule, the kit comprising:

(a) a solid support, the surface of which is activated with a leaving group to facilitate non-covalent attachment of the DNA molecule to the solid support; and

(b) instructions for determining the methylation status of the one or more cytosine residues. s 28. A kit for bisulphite treatment of DNA, the kit comprising:

(a) a solid support and at least one linker to facilitate attachment of at least one end of a DNA molecule to the solid support; and

(b) instructions for treating the DNA molecule with sodium bisulphite to modify the one or more cytosine residues.