WO2007033834A2

WO2007033834A2 - Method for the analysis of nucleic acid methylation

Info

Publication number: WO2007033834A2
Application number: PCT/EP2006/009233
Authority: WO
Inventors: Sonja Sievers; Oliver MÜLLER
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 2005-09-23
Filing date: 2006-09-22
Publication date: 2007-03-29
Anticipated expiration: 2008-03-23
Also published as: WO2007033834A3

Abstract

The present invention relates to a method for the analysis of nucleic acid methylation.

Description

Method for the Analysis of Nucleic Acid Methylation

Description

So far available methods for the analysis of methylation of nucleic acids are non-quantitative, laborious or expensive. Examples of available techniques are methylation specific PCR (MSP) and methylation sensitive restriction hydrolysis followed by PCR (Feinberg, A.P. and Vogelstein B., Nature 301 (1983) 89-92; J. Singer-Sam et al., Nucleic Acid Res. 8 (1990) 687; J.G. Herman et al., Proc. Natl. Acad. Sci. USA 93 (1996) 9821-9826). Further disadvantages of known methods are the high amount of DNA which is required and the impossibility of automatisation, which, however, is essential for the analysis of a high number of samples. A further method called methylation-sensitive single-nucleotide primer extension (ms-SNuPE) is based on the detection of radioactively or fluorescently labelled nucleotides (M.L. Gonzalgo et al., Methods 27 (2002) 128-133).

An object of the present invention was therefore to provide an improved method for the analysis of methylation, in particular a method which allows for fast and easy quantification of the proportion of methylated and non- methylated nucleic acids in a given sample. This object is solved according to the invention by a method for the analysis of nucleic acid methylation comprising the steps:

a) providing a sample comprising a nucleic acid to be analyzed, b) modifying the nucleic acid in such a way that non-methylated nucleotides having a predetermined nucleobase are converted or that methylated nucleotides having the predetermined nucleobase are converted, c) performing a first amplification reaction of the nucleic acid to be analyzed, which amplification reaction is non-specific for methylation, d) performing a second amplification reaction being a solid phase amplification reaction comprising a first immobilized primer which is non-specific for methylation and a second non-immobilized methylation-specific primer having a marker group, and e) analyzing nucleic acid methylation by detection of the amplification product of the second amplification reaction containing the marker group.

According to the invention, a fast, easy and low cost method for the analysis, in particular also for the quantitative analysis of nucleic acid methylation, in particular DNA methylation is provided which can be automated.

According to the invention, in a first step (a), a nucleic acid to be analyzed is provided, which nucleic acid, in particular, is a methylated nucleic acid. Preferably, the nucleic acid is DNA and most preferably genomic DNA. The nucleic acid to be analyzed preferably is a human genomic DNA which is methylated. In human cells the motif CpG is methylated. For a positive control an in vitro methylated nucleic acid can be used.

Methylation can be performed by treating the nucleic acid with an enzyme, in particular with a methyl transferase. Suitable methyl transferases include M.Sssl, Dnmti human DNA (cytosine-5) methyltransferase, AIu I methylase, BamH I methylase/, dam/methylase, EcoR I methylase, Hae III methylase, Hha I methylase, Hpa Il methylase, Msp I methylase and Taq I methylase. Particularly preferred, M.Sssl is used. This methyl transferase specifically methylates cytosine nucleotides within the motif CpG and is therefore suitable for methylation and analysis of human DNA. Other enzymes are used for analysis of bacterial, animal or plant DNA, depending on the respective methylation motifs.

In a next step b), specifically methylated nucleic acid or non-methylated nucleic acid are modified. Modification includes conversion of methylated nucleotides or non-methylated nucleotides into other chemical entities, which lateron allow for distinction between methylation and non-methylated species. Preferably, all non-methylated cytosines are modified and all methylated cytosines are not converted. Particularly preferred, the nucleic acid, in particular genomic DNA is modified using sodium bisulfite. Such procedure is e.g. described by M. Frommer et al., Proc. Natl. Acad. Sci. USA 89 (1992), 1827-1831. Using sodium bisulfite non-methylated cytosine nucleotides are modified to uracil nucleotides, while methylated cytosine nucleotide are not converted.

In step (c), a first amplification reaction is performed, which amplification reaction is non-specific for methylation. Non-specific for methylation means that both methylated and non-methylated nucleic acids or modified and non- modified nucleic acids, respectively, are amplified. Particular preferred, the first amplification reaction is a polymerase chain reaction (PCR). To obtain a methylation unspecific amplification, primers are used which anneal independently from previous methylation to the DNA or DNA fragment to be analyzed, e.g. to a fragment of a genomic template. In the first amplification reaction a first amplification product is obtained.

In a particularly preferred embodiment, in which a modification using sodium bisulfites was performed, cytosines which have not been methylated and were therefore converted to uracils are now replicated as thymine nucleotides. Thus, originally non-methylated C, e.g. non-methylated CpG pairs, are transferred into T, in particular into TpG pairs by bisulfite treatment and following the first amplification reaction, in particular a PCR.

On the other side, originally methylated C, e.g. CpG pairs are staying unchanged and are replicated as C, e.g. CpG pairs by this non-methylation specific first amplification reaction.

The amplification product of the first amplification reaction then serves as template in a second amplification reaction in step (d) of the present invention.

The second amplification reaction is a solid phase amplification reaction, in particular a solid-phase PCR using a first immobilized primer, which is non- specific for methylation and a second non-immobilized methylation-specific primer. Preferably, the first methylation unspecific primer is covalently bound to a solid phase and thus immobilized. Examples of a suitable solid phase are: a wall of a reaction tube, which can be a single tube or a well of a Multiwell plate, a magnetic or non-magnetic microbead, a complex of several macromolecules (nanobead), a carbohydrate-based or polymer-based membrane.

Using a second non-immobilized methylation-specific primer, the sequence is amplified. The second primer only anneals to sequences which were originally methylated or only to sequences which were originally non- methylated. Further, the second primer comprises a marker group which allows for detection and in particular for quantitative detection. Suitable marker groups include e.g. a part of a specific binding pair, such as biotin, avidin or streptavidin, a direct label such as a colour label, a label which is detectable at fluorescent, UV, visible or infrared light, a radioactive label, a protein label, e.g. green fluorescent protein (GFP), or a directly bound or indirectly bound enzyme label such as horseradish peroxidase (HRP), alkaline phosphatase, luciferase. The analysis, in particular the quantitative analysis can then be performed using the marker group. In a particular preferred embodiment, the marker group is biotin, e.g. a biotinylated second primer is used. For detection, then streptavidin can be added, which binds to the immobilized biotinylated amplification product. Next a biotinylated enzyme, e.g. biotinylated horse-radish peroxidase (HRP) or another reporter enzyme is added which binds to the streptavidin. Quantification can then be performed by enzymatic colorimetric detection, e.g. by adding TMB as colorless substrate, which is converted into a blue substrate in a colorimetric enzyme reaction. In a particular preferred embodiment, the number of cycles in the first amplification and/or the second amplification reaction is ≤ 50, in particular ≤ 35 and more preferred ≤ 30. Using a low number of amplification cycles, in particular in the second amplification reaction results in that the amount of amplification product correlates linear with the amount of templates. Thus, a fast and immediate quantification can be made.

In a further preferred embodiment of the invention, the second amplification reaction is performed using an additional third primer. Further, one of the second and third primer is annealing specifically to the methylated sequence, while the other is specifically annealing to non-methylating sequence. By this embodiment, it is possible to directly determine the proportion of methylated to non-methylated nucleic acid indepedently of the number of cycles of the second amplification reaction. Thus, in this embodiment, a high number of cycles, in particular, < 50, more preferred < 100 can be used.

The invention is further explained by the following Examples and the Figures wherein Figure 1 schematically shows steps (d) and (e) of the present invention. DNA is modifed with bisulfite and amplified in PCR which is unspecific for methylation (not shown). After immobilization of the primer, the colorimetric detection is made after solid phase (PCR) binding of streptavidin and biotinylated HRP and the addition of TMB.

Figure 2 shows the specificity of the inventive method. Shown is the detection of the product of a solid-phase of a PCR, whereby the product is indicated by yellow color.

Figure 3 shows quantification of the proportion of methylated DNA. Known percentages of methylated DNA are shown. The columns indicate the absorptions of the samples. The results are mean values from two measurements. Examples

Example 1

Analysis of the promoter methylation of the APC gene

Step 1. DNA isolation

Genomic DNA is isolated from human embryonic kidney 293 (HEK293) cells using standard method

(QiaAmp DNA Mini Kit, Qiagen).

Step 2. Making the positive control

An aliquot of the purified DNA is enzymaticaDy methylated. For this 10 μg DNA are incubated in 5( μ] methylation buffer (16 U M.SssT in 50 mM NaCl, 10 mM Tris/HCl (pH 7.4), 10 mM MgCl., 1 mlv

Dithiothreitol, 160 μM S-adenosylmethionine) for 2 h at 37⁰C.

Step 3. Bisulfite modification

The bisulfite modification of 1 μg DNA starts with an incubation in 50 μl denaturing buffer (300 mM

NaOH) at 42⁰C for 20 min. NeJrt the DNA is deaminated in 4 M sodium bisulfite and 10 mM h.'άrochinone for 4 h at 55°C. The DNA is desalted using the Qiagen PCR purification kit and desulfonated in denaturins buffer at 37⁰C for 15 min.

Step 4. First PCR (methylation unspecific)

In this first methylation unspecific PCR primers are used, which anneal independently of a

ious mεthylation to the fragment of the genomic DNA template. Primer A 5'- GGGTTGTATTAATATAGTTATAT-3' is complementary to the sequence 829 through 851 of the promoters of the APC gene (Gene Database U02509.1). Primer B 5'- TTCCTTACTTACTAAAAACTAAAA-S' is complementary to the promoter sequence from position 600 through 628. The PCR is performed with 600 nM primer A, 600 nM primer B in 25 μl PCR buffer (Ix Qiagen PCR Puffer, 200 μM dNTP, 0,5 U/ml Hot Start Taq Polymerase (Qiagen)) in 30 cycles. One cycle consists of incubations at 94⁰C for 30 s, at 50⁰C for 30 s and at 72⁰C for 30 s. Step 5. Covalent binding of the 5' primer to the solid phase

The phosphorylated primer C S'-P-TTTTTTTTTTGGGTTGTATTAATATAGTTATAT-S' is coupled to the activated solid phase θ£ a Nucleolink Strip (Nunc). For this the well is incubated with 100 μ\ primer binding buffer (5 μM primer C, 10 mM methylimidazol, 10 mM l-ethy]-3 -(3 -dimethyl - aminopropyl)-carbodiimid) for 8 h at 50⁰C. The sequence of primer C is identical to the sequence of primer A with additional 10 thymine nucleotides at the 5'-end. Step 6. Second solid-phase PCR (methylation specific)

The second PCR is performed with 500 nM primer D and 62.5 nM primer C in 50 μ\ PCR buffer. The PCR is done in 25 cycles, when one cycle consists of incubations at 94⁰C for 30 s, at 50°C for 30 s and at 72°C for 30 s. The 5'-end biotinylated primer D 5'B-CGAAATACGAATCGAAAAACG-S ' is complementary to the sequence 707 to 727 of the promoter of the APC gene. Primer D anneals only to DNA, which was originally non-methylated. As second primer the immobilized primer C is used. Step 7. Washing and detection

The PCR reaction solution is removed. 200^~/H^"washing buffer (100 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1 % Tween 20) are added and removed. The well is incubated for 5 min in 200 μl washing buffer. Washing is made three times Then 100 μ\ detection buffer 1 (100 mM Tris/HCl (pH 7.5), 150 mM Na^Q, 0.1 % Tween 20, 0.1 μglπύ strεptavidin, 25 μ°l\ biotin-HRP) are added, incubated for 1 h at 37°C and the wells are washed with washing buffer. 100 μ\ detection buffer 2 (1.2 mM tetramethylbenzidine) are added and incubated for 25 min at 20⁰C. The reaction is stopped by addition of 100 μ\ stopping solution (100 mM H₂SO₄). The detection is performed by measuring the absorption in a plate reader at 450 nm. Example 2

Test of Specificity

The specificity of the methylation specific PCR and the following detection was tested by comparing analysis of methylated and non-methylated DNA as shown in Figure 2.

Example 3

Quantification

The portion of methylated DNA was tested by analysis of mixtures of known percentages of methylated and non-methylated DNA as shown in Figure 3.

Claims

1. A method for the analysis of nucleic acid methylation comprising the steps:

a) providing a sample comprising a nucleic acid to be analyzed, b) modifying the nucleic acid in such a way that non-methylated nucleotides having a predetermined nucleobase are converted or that methylated nucleotides having the predetermined nucleobase are converted, c) performing a first amplification reaction of the nucleic acid to be analyzed, which amplification reaction is non-specific for methylation, d) performing a second amplification reaction being a solid phase amplification reaction comprising a first immobilized primer which is non-specific for methylation and a second non-immobilized methylation-specific primer having a marker group, and e) analyzing nucleic acid methylation by detection of amplification product of the second amplification reaction containing the marker group.

2. The method of claim 1 , wherein the nucleic acid is DNA.

3. The method according to any of the preceding claims, wherein the nucleic acid is a genomic DNA.

4. The method of any of claims 1 to 3, for the quantitative analysis of methylation.

5. The method according to any of the preceding claims, wherein the first amplification reaction and/or the second amplification reaction is a polymerase chain reaction (PCR).

6. The method according to any of the preceding claims, wherein the marker group contained in the non-immobilized methylation-specific primer is a partner of a specific binding pair, a direct label or an enzyme label.

7. The method according to any of the preceding claims, wherein the number of cycles in the first amplification reaction and/or the second amplification reaction is < 50.