DE10347400B4 - Bisulfite conversion of DNA, involves reacting genomic DNA with bisulfite reagent, in presence of compound out of group of dioxane, one of its derivatives and similar aliphatic cyclic ether - Google Patents

Abstract

Bisulfite conversion (M1) of DNA, involves reacting genomic DNA with a bisulfite reagent, where the reaction is carried out in the presence of a compound out of the group of dioxane, one of its derivatives and a similar aliphatic cyclic ether. - Bisulfite conversion (M1) of DNA, involves reacting genomic DNA with a bisulfite reagent, where the reaction is carried out in the presence of (a) a compound out of the group of dioxane, one of its derivatives and a similar aliphatic cyclic ether, (b) a compound of formula called (1), or (c) the reaction is conducted at a temperature in the range of 0-80 deg. C and that the reaction temperature is increased to a range of 85-100 deg. C briefly during the course of the conversion (thermospike). - n = 1-35000; - m = 1-3;and - R1,R2 = H, Me, Et, Pr or Bu. - An INDEPENDENT CLAIM is also included for a kit, comprising a reagent containing bisulfite, denaturing reagents or solvents, and scavengers and primers for the production of amplificates, and optionally an ultrafiltration tube or instructions for performing (M1).

Description

background the invention

The The present invention relates to a method for the analysis of cytosine methylations in DNA. 5-methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. She plays an important biological Role, i.a. in transcriptional regulation, in genetic imprinting and in tumorigenesis (for overview: Millar et al .: Five not four: history and significance of the fifth base. In: S. Beck and A. Olek, eds .: The Epigenome. Wiley-VCH publishing house Weinheim 2003, pp. 3-20). The identification of 5-methylcytosine as a component of genetic Information is therefore of considerable interest. A proof of However, methylation is difficult because cytosine and 5-methylcytosine have the same base pairing behavior. Many of the conventional, Hybridization based detection methods are therefore capable not to differentiate between cytosine and methylcytosine. In addition, goes completely lost the methylation information in a PCR amplification.

The conventional methods for methylation analysis operate essentially on two different principles. On the one hand methylation-specific restriction enzymes are used, on the other hand there is a selective chemical conversion of non-methylated cytosines into uracil (so-called: bisulfite treatment, see for example: DE 101 54 317 A1 ; DE 100 29 915 A1 ). The enzymatically or chemically pretreated DNA is then usually amplified and can be analyzed in various ways (for a review: WO 02/072880 p. 1 ff; Fraga and Esteller: DNA Methylation: A Profile of Methods and Applications, Biotechniques 33: 632-649 , Sept. 2002).

Since the treatment with methylation-specific restriction enzymes is restricted to specific sequences by the sequence specificity of the enzymes, bisulfite treatment is carried out for most applications (for a review) DE 100 29 915 A1 5.2, lines 35-46).

The Bisulfite treatment is classically carried out in the following steps: The genomic DNA is isolated by shearing or by treatment with Restriction enzymes fragmented, denatured with sodium hydroxide, reacted with a concentrated bisulfite solution for several hours and subsequently desulfonated and desalted (see: Frommer et al .: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1; 89 (5): 1827-31).

Recently several technical improvements of this method have been developed. Thus, the DNA to be examined in the so-called agarose bead method is enclosed in an agarose matrix. This prevents diffusion and renaturation of the DNA. All precipitation and purification steps are then replaced by rapid dialysis. With this method it is possible to study the methylation status of individual cells. However, there are difficulties with very small fragments that are lost by diffusion (Olek et al .: A modified and improved method for bisulphite-based cytosine methylation analysis, Nucleic Acids Res. 1996 Dec 15; 24 (24): 5064-6.) , In the patent application DE 100 29 915 A1 (WO 01/98528) is a bisulfite method described in which a DNA sample with a bisulfite solution in the concentration range of 0.1 mol / l to 6 mol / l in the presence of a denaturing reagent and / or solvent and at least a radical scavenger is incubated. Many different suitable denaturing substances and radical scavengers are described in this patent application. In the patent application DE 101 54 317 (WO 03/038121) discloses a method in which the DNA to be examined is bound to a surface during the bisulfite treatment, thereby simplifying purification and washing steps.

A fundamental problem with bisulfite treatment, however, is that long reaction times are required to ensure complete conversion, thus eliminating false-positive results, for example. At the same time, however, the long reaction times lead to a degradation of the DNA. Although higher reaction temperatures lead to a higher conversion rate, but also to a greater degradation of the DNA. The interactions between temperature, reaction time, conversion and degradation rates have recently been systematically investigated. It could be shown that the highest conversion rates are achieved at temperatures of 55 ° C (with reaction times between 4 and 18 hours) and at 95 ° C (with a reaction time of one hour). A big problem, however, is the degradation of DNA. Thus, 84-96% of the DNA is degraded at a reaction temperature of 55 ° C. Degradation is even higher at 95 ° C (Grunau et al .: Bisulfite genomic sequencing: systematic investigation of critical experimental parameters, Nucleic Acids Res. 2001 Jul 1; 29 (13): E65-5). Accordingly, most authors use reaction temperatures of about 50 ° C (see: Frommer et al., Aa, 1992, p 1827, Olek et al., 1996, p 5065, Raizis et al: A bisulfite method of 5-methylcytosine mapping that minimizes template degradation: Anal Biochem., 1995 Mar 20; 226 (1): 161-6, 162). In addition to the high rate of degradation of DNA is another problem of the conventional bisulfite method in that a powerful purification method of the converted DNA has not been described so far. Many authors use precipitations (see: Grunau et al., Aao). Purification via DNA-binding surfaces has also been described (cf .: Kawakami et al .: Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma Journal of the National Cancer Institute, Vol 92, No. 22, 2000, pp. 1805 -11). The yield of these purifications is limited.

The DE 101 32 211 A1 describes detection of specific dinucleotides in DNA samples by fluorescence resonance energy transfer (FRET).

Furthermore, the describes DE 197 54 482 A1 a method for producing complex DNA methylation fingerprints. There is described a three-step process. The first step is deamination (at high temperature). As a second step - between deamination and renewed sulphonation - denaturation is now introduced (temperature> 90 ° C). In the third step, the sulphonation (low temperature) takes place. The individual steps (temperature steps) are in each case carried out under equilibrating conditions with formation of platelets. These are each not short-term increases in temperature returning to the previous temperature level.

By the high losses of conventional Bisulfite treatment, it is problematic, these procedures for investigations use, in which limits the amount of DNA to be analyzed is. A particularly interesting field of application of methylation analysis lies precisely in it, using DNA from body fluids, such as blood or urine, Cancers or others with a change in the methylation status to diagnose associated diseases. In bodily fluids, however, the DNA comes only in low concentrations, so that the applicability of the Methylation analysis by the low yield of conventional Bisulfite treatment limited is.

Therefore exists because of the particular importance of cytosine methylation and because of the mentioned Disadvantages of conventional methodology a great technical need improved process for bisulfite conversion.

It was now a surprise found effective way in which the conversion rate of the bisulfite reaction unexpectedly increased significantly can be while at the same time the required reaction time and thus also the Degradation rate of DNA is significantly reduced. Here is the Reaction temperature of bisulfite conversion in the course of the reaction increased for a short time. By combining the temperature increases with new solvents and new purification methods can reduce the efficiency of Implementation in addition increase significantly. A sensitive methylation analysis of tissue or from body fluids isolated DNA is possible.

description

at the method according to the invention For example, bisulfite conversion is carried out at mild reaction temperatures (0-80 ° C). in the During the reaction, the reaction temperature then becomes at least once increased significantly for a short time. These short-term temperature increases are referred to below as "thermospikes." The "normal" reaction temperature outside the thermospike is called the reaction base temperature.

Therefore the invention is a bisulfite conversion process DNA, wherein the reaction base temperature is between 0 ° C and 80 ° C, and the reaction temperature during the reaction at least once briefly over 85 ° C is increased.

The DNA to be examined may vary depending on diagnostic or scientific Question from different sources. For diagnostic Studies serve as starting material, preferably tissue samples, but also body fluids, especially serum. Possible is also the DNA from sputum, stool, urine or cerebrospinal fluid to use. Preferably, the DNA is from the biological samples isolated. The DNA extraction is done according to standard methods, from blood using the Qiagen UltraSens DNA Extraction Kits. Other methods of purifying DNA are known to those skilled in the art.

Then you can the isolated DNA may be fragmented by, for example, turnover with restriction enzymes. The reaction conditions and the enzymes of interest are known in the art and arise, for example, from the manufacturer supplied Protocols.

The bisulfite conversion can be carried out according to the known protocols given above. The reaction can take place both in solution and on a solid phase (cf. DE 100 29 915 ; DE 101 54 317 ). Preferably, sodium disulfite (= sodium bisulfite / sodium metabisulfite) is used because it has a higher water solubility than sodium sulfite. The disulfite salt disproportionates in aqueous solution to the hydrogen sulfite anions required for the cytosine conversion. Is spoken in the following of bisulfite, so be this is due to the concentration of hydrogen sulfite and sulfite anions in the reaction solution. Concentration ranges of 0.1 to 6 mol / l are possible for the process according to the invention (cf. DE 100 29 915 ). Particularly preferred is a concentration range of 1-6 mol / l, most preferably 2-4 mol / l. When using certain solvents, however, the maximum usable concentration of bisulfite may be lower (see below). When choosing the bisulfite concentration, it should be taken into account that a high concentration of bisulfite leads to a high conversion, but due to the lower pH also leads to a high degradation rate

In a preferred embodiment, the reaction takes place in the presence of a radical scavenger and a denaturing reagent or a corresponding solvent (cf. DE 10029 915 ). In this case, different radical scavengers known to those skilled in the art can be used (cf. DE 100 29 915 ). Chroman derivatives are more preferably used, such as 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. In a particularly preferred embodiment, the denaturing solvent used is dioxane, one of its derivatives or a similar aliphatic cyclic ether. Dioxane is present in the reaction mixture preferably in a concentration of 10 to 35 vol.% Before. A higher dioxane content than 35% is problematic because it then comes to a two-phase formation in the reaction mixture. Particularly preferred is a Dioxankonzentration of 20-30%, in particular 22-28%, wherein the bisulfite concentration between 3.3 and 3.6 mol / l. Very particular preference is given to a dioxane content of 25% at a bisulfite concentration of 3.5 mol / l.

wobei
n = 1–35000
m = 1–3
R1 = H, Me, Et, Pr, Bu
R2 = H, Me, Et, Pr, Bu
ist.In another particularly preferred embodiment, compounds of the following formula are used as denaturing solvents:

in which
n = 1-35000
m = 1-3
R1 = H, Me, Et, Pr, Bu
R2 = H, Me, Et, Pr, Bu
is.

Especially Preference is given to n-alkylene glycol compounds, in particular their dialkyl ethers, in particular again diethylene glycol dimethyl ether (DME).

The Compounds of the invention can be used in different concentrations. DME is preferred in concentrations between 1-35% used. Preference is given to 5 to 25%, particularly preferably 10% DME. The bisulfite concentration is preferably between 0.1-6 mol / l, especially preferably between 1-6 mol / l, most preferably between 3-4.5 mol / l.

The Bisulfite conversion can be carried out in a wide temperature range (so.). The process according to the invention uses reaction base temperatures from 0 to 80 ° C. When choosing the basic reaction temperature is to be considered that a higher temperature not only to speed up the transformation, but also leads to an increase in the rate of degradation. In a preferred embodiment of the invention, the reaction temperature is basic therefore between 30-70 ° C. Especially preferred is a range between 45-60 ° C; very particularly preferred are 50-55 ° C.

The optimal reaction time of the bisulfite treatment depends on the reaction temperature from. The reaction time is usually between 1 and 18 hours (see: Grunau et al supra). For a reaction temperature of 50 ° C the reaction time is usually at 4-6 Hours. If you work with Thermospikes, you can do the required Reduce reaction time, however (see examples).

The optimal number of thermospikes depends on the reaction temperature. there the lower the reaction base temperature, the higher the optimal number of thermospikes is. In any case, at least one Thermospike is required. On the other hand, there are basically any number of thermospikes conceivable. To be considered However, that is when there are a large number of temperature increases as well the rate of degradation of DNA increases, and thus optimal implementation no longer guaranteed is. The preferred number of thermospikes is therefore depending on Reaction base temperature between 1 and 10 thermospikes. Especially 2 to 5 thermospikes are preferred.

The Increase thermospikes the reaction temperature is more preferably 85 to 100 ° C, more preferably 90 to 98 ° C preferably at 94 ° C-96 ° C.

The Duration of temperature increases also depends from the volumes of the reaction mixtures. It must be guaranteed be that the temperature is increased evenly throughout the reaction solution. When using a thermocycler for a 20 ul reaction mixture 30 seconds temperature increase is sufficient. At a volume of 100 μl are 1.5 minutes and at 600 μl 3 minutes temperature increase required.

Upon completion of the bisulfite conversion, desulfonation and purification of the DNA occurs. For this purpose, different methods are known (see, for example: DE 101 54 317 A1 ; DE 100 29 915 A1 ; Grunau et al. 2001, aao). Normally, the reaction solution is first treated with sodium hydroxide solution. This is followed by neutralization and alcohol precipitation of the DNA.

According to the invention preferred The purification is done by means of a gel filtration, about with Sephadex G25 columns. Herewith The bisulfite salt can be removed very effectively without further washing steps would be required. In a second preferred embodiment, purification is via DNA-binding Surfaces, about that Wizard DNA Purification Resin from Promega (Cf.: Kawakami et al. A.o.). Information on the purification of nucleic acids by gel filtration or DNA-binding surfaces are known in the art and arise approximately from the manufacturer's information. In a particularly preferred embodiment, the purification is carried out by ultrafiltration. Such a procedure has several technical advantages and leads to a surprising effective purification. So the recovery rate is the converted DNA very high (> 85%). This applies to both high molecular weight DNA as well as for Fragmented DNA, as occurs in body fluids. The conventional ones On the other hand, processes for isolating bisulfite-treated DNA lead only to a recovery rate of about 25%. The ultrafiltration also has other benefits. So is the purification regarding the Volumes of the samples to be used very flexible. In addition, the Bisulfite salts almost completely be removed. Last but not least is desulfonation on the filter membrane possible, what else leads to a time saving.

the Those skilled in the art are various commercially available ultrafiltration systems known for the inventive method can be used. In a preferred embodiment become the Microcon columns used by Millipore. The purification can be carried out after a modified Manufacturer's log. For this purpose, the bisulfite reaction solution with Water and added to the ultrafiltration membrane. After that will the reaction solution for about Centrifuged for 15 minutes and then with 1 × TE buffer (mixture of 10 mol / l Tris and 0.1 mol / l EDTA with a pH of 8). The DNA remains on the membrane during this treatment. Then done the desulfonation. For this purpose, 0.2 mol / l NaOH is added and for 10 min incubated. Subsequently Another centrifugation (10 min) and a washing step with 1 × TE buffer. Subsequently the DNA is eluted. For this purpose, the membrane is warm for 10 minutes with 50 .mu.l 1 × TE buffer (50 ° C). The Membrane is turned according to manufacturer's instructions and there is a re-centrifugation to remove the DNA from the membrane becomes. The eluate can be used directly for the detection reactions are used. The person skilled in the art is aware that other approaches appear in other ultrafiltration systems could be, and that a good yield even with variation of the above Conditions can be achieved. The corresponding embodiments are also part of this invention.

In In another particularly preferred embodiment, the purification is carried out using magnetic particles, such as the Magna-Pure process (Roche). This purification method leads in particular to DME and the others mentioned above Links to particularly good results. The purification takes place essentially according to the manufacturer's instructions. The person skilled in the art is aware that with variation of the manufacturer's instructions by standard tests an increased Yield can be achieved. Correspondingly optimized protocols are also part of this invention.

The converted and purified DNA can be analyzed by different routes. For this purpose, a variety of possible methods are known in the art (for review: Fraga and Esteller 2002, pp. 634, 641 et seq.). It is particularly preferred to first amplify the DNA by means of a polymerase chain reaction. Various methods can be used to ensure selective amplification of the originally methylated or unmethylated DNA, for example via the so-called "heavy-methyl" method (for review: WO 02/072880) or the so-called "methylation-sensitive PCR"("MSP See: Herman et al .: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands Proc Natl Acad Sci USA 1996 Sep 3; 93 (18): 9821-6). The detection of the amplificates can be carried out by conventional methods, for example via primer extension reactions ("MsSNuPE"; DE 100 10 280 or by hybridization to oligomer arrays (See, for example: Adorjan et al., Tumor class prediction and discovery by microarray-based DNA methylation analysis., Nucleic Acids Res. 2002 Mar 1; 30 (5): e21). In another particularly preferred embodiment, the amplificates are analyzed using PCR real-time variants (cf. US 6,331,393 "Methyl-Light"). Preferred variants are the "Taqman" and the "Lightcycler" process.

Another aspect of the invention is the use of all embodiments of the invention. If disease-specific cytosine positions are investigated, the method according to the invention is particularly suitable for the diagnosis or prognosis of cancers or other diseases associated with a change in the methylation status. These include CNS miss functions, symptoms of aggression or behavioral disorders; clinical, psychological and social consequences of brain damage; psychotic disorders and personality disorders; Dementia and / or associated syndromes; cardiovascular disease, dysfunction and damage; Dysfunction, damage or disease of the gastrointestinal tract; Dysfunction, damage or disease of the respiratory system; Injury, inflammation, infection, immunity and / or convalescence; Dysfunction, damage or disease of the body as a deviation in the development process; Dysfunction, damage or disease of the skin, muscles, connective tissue or bones; endocrine and metabolic dysfunction, injury or disease; Headache or sexual dysfunction. The method according to the invention is also suitable for predicting adverse drug reactions, establishing a specific drug therapy (personalized medicine) and monitoring the success of a drug therapy. Another application is the differentiation of cell types or tissues and the study of cell differentiation.

example 1

Bisulfite conversion with the help of thermospikes

To 1 μl of MssI digested, high purity human DNA (Promega, 160 ng) was added 2 μl of ddH ₂ O. The sample was denatured for 10 minutes at 96 ° C. Subsequently, 10 μl of bisulfite solution (5.85 mol / l) and 7 μl of a scavenger-dioxane mixture (5 μl of dioxane plus 2 μl of radical scavenger) were added rapidly. Subsequently, the first sample (0 h value) was removed and placed on ice. The reaction mixture was incubated for 30 seconds at 96 ° C and then for 59.5 minutes at 50 ° C. The second sample (1 h value) was removed and placed on ice. The third sample (2 h value) was incubated again at 96 ° C for 30 seconds and at 50 ° C for 59.5 minutes. Subsequently, this sample was also put on ice. The fourth sample (3 h value) was incubated once again for 30 seconds at 96 ° C and for 59.5 minutes at 50 ° C and then also cooled. To the samples was added 30 μl of ddH ₂ O. The reaction mixture was purified on G25 Sephadex columns. The eluate was admixed with 50 μl of 100 mM Tris-HCl (pH 9.5) and desulfonated at 96 ° C for 20 minutes. 2 μl of this solution was used for each PCR reaction. Two bisulfite-specific fragments, two nonspecific fragments and one genomic fragment were amplified in the PCR. The bisulfite-specific fragments are the more amplified the further the bisulfite conversion has progressed. The non-specific fragments are amplified independently of the bisulfite conversion and give an indication of the degradation of the DNA. The genomic fragment is amplified only to the extent that genomic, non-bisulfite converted DNA is still present. The amplification of genomic DNA is therefore a measure of incomplete bisulfite conversion. The amplifcates separated in agarose gels are in 1 to see. It turns out that in the process according to the invention, a large part of the DNA is already reacted after one hour. At the latest after three hours no genomic DNA is detectable, ie the bisulfite conversion is complete. In the case of the conventional bisulfite treatment, corresponding values result at the earliest after 5 h (see below).

Example 2

Comparison of bisulfite treatment with thermospikes opposite bisulfite treatment without thermospikes

As in Example 1 treated samples were incubated at a reaction time of 3 or 5 h with two thermospikes. The controls were implemented without Thermospikes. Purification and PCR were as described above. Two bisulfite-specific fragments were amplified. One of the fragments is cytosine-rich. Therefore, a relatively long reaction time is required to achieve complete conversion. The other fragment, on the other hand, is cytosine-poor and therefore completely converted after a relatively short time. The results of the amplifications are in 2 shown. On the left images you can see the conventional bisulfite conversion, on the right images the optimized conversion with termospikes. The cytosine-rich fragment is on the left side, the cytosine-poor fragment on the right side. It turns out that the method according to the invention enables a much more sensitive detection. Thus, the cytosine-rich fragment is clearly detectable in a Thermospikes treatment after only 3 hours, while the conventional method even after 5 hours of reaction time is not capable of detection.

Example 3: Optimized Bisulfite conversion for the detection of DNA in plasma samples.

It should be shown that the optimized bisulfite method allows a sensitive methylation analysis of body fluid derived DNA. For this purpose, 1 ml of human plasma was mixed with a certain amount of human DNA. The DNA was isolated from the plasma samples via the Magna Pure method (Roche) according to the manufacturer's instructions. The resulting from the purification 100 .mu.l eluate were completely used in the following bisulfite reaction. This was done as a control the Reaction according to a standard procedure (Frommer et al., Aao). The procedure according to the invention was as follows: The eluate was treated with 354 μl bisulfite solution (5.89 mol / l) and 46 μl DME (including radical scavenger (6-hydroxy-2,5,7,8-tetramethylchroman-2-one) carboxylic acid, 98.6 mg in 787 μl of DME) The reaction mixture was denatured for 3 minutes at 99 ° C. and then incubated for a total of 5 hours in the following temperature program: 50 ° C. for 30 minutes, one thermospike (99.9 ° C.) for 3 minutes, 1.5 hours at 50 ° C., a thermospike (99.9 ° C.) for 3 minutes, and 3 hours at 50 ° C. The reaction mixtures of both the control and the process according to the invention were subsequently subjected to ultrafiltration by means of a Millipore microcon Purification was carried out essentially according to the manufacturer's instructions: 300 μl of water were added to the reaction mixture, added to the ultafiltration membrane, centrifuged off for 15 minutes and then washed with 1 × TE buffer, the DNA remaining on the membrane during this treatment Conn The desulfonation takes place. To this was added 0.2 mol / l NaOH and incubated for 10 min.

This was followed by centrifugation (10 min) and a washing step with 1 × TE buffer. Thereafter, the DNA was eluted. For this purpose, the membrane was mixed for 10 minutes with 50 μl of warm 1 × TE buffer (50 ° C.). The membrane was turned according to manufacturer's instructions. This was followed by another centrifugation, with which the DNA was removed from the membrane. 10 μl of the eluate were used for the following Lightcycler Real-Time PCR. In this case, the amplification was carried out by means of a bisulfite-specific assay. The results calculated by the Lightcycler software are in 3 shown. The left curves correspond to the optimized method, the right curves to the conventional method. It turns out that the optimized method generates a significant fluorescence signal even at low number of cycles. The DNA yield is therefore higher than in the conventional method. By comparison with calibration curves, the amount of converted DNA can be quantified. The optimized procedure resulted in a DNA concentration of 133.27 ng / 100 μl, while the conventional method resulted in a concentration of 41.03 ng / 100 μl. The process according to the invention therefore enabled a threefold higher yield than the conventional process.

description the figures

1

1 shows the results of Example 1. Shown is the gel electrophores after a PCR amplification. Two different bisulfite-specific fragments, two nonspecific fragments and one genomic fragment were amplified. The top picture shows the zero value (reaction time = 0 hours). The second image from the top shows the reaction with a thermospike and an hour of reaction time. The third picture from the top corresponds to the reaction with two thermospikes and two hours reaction time. The bottom picture shows a reaction with three thermospikes and three hours reaction time. With the method according to the invention, a large part of the DNA is already reacted after one hour (second image from above). At the latest after three hours, no genomic DNA is detectable (bottom).

2

2 shows the results of Example 2. Shown is the gel electrophores after a PCR amplification. In each case two different, bisulfite-specific amplicons were amplified. The left-hand pictures show conventional bisulfite treatments, the right ones the process according to the invention. Above is a reaction time of 3h, below a reaction time of 5 h. With the method according to the invention (thermospikes) a significantly more sensitive detection is possible. Thus, the fragment applied in the left lane is already detectable after 3 reaction time, while the conventional method is not able to detect the fragment even after 5 hours of incubation.

3

3 shows the results of Example 3. DNA was isolated from plasma samples, bisulfite treated and amplified by a Lightcycler PCR. The Y axis shows the fluorescence signal measured in each cycle. The X-axis indicates the number of cycles. On the left side of the curves of the method according to the invention are shown, on the right side of the conventional method. It turns out that the optimized method generates a significant fluorescence signal even at low number of cycles. The DNA yield is higher than in the conventional method.

Claims (22)

A process for bisulfite conversion of DNA, characterized in that the reaction base temperature is 0 ° C to 80 ° C and that the reaction temperature in the course of the reaction at least once briefly, Thermospikes, is increased to above 85 ° C. Method according to claim 1, characterized in that that the bisulfite conversion in the presence of a denaturing Reagent is performed. A method according to claim 2, characterized gekenn indicates that the denaturing reagent is dioxane, one of its derivatives, or a similar aliphatic cyclic ether. Method according to claim 3, characterized that the concentration of dioxane is 10-35%. A method according to claim 2, characterized in that the denaturing reagent is a compound having the formula:

where n = 1-35000 m = 1-3 R1 = H, Me, Et, Pr, Bu R2 = H, Me, Et, Pr, Bu. Method according to claim 5, characterized that the denaturing reagent is an n-alkylene glycol compound is. Method according to Claim 6, characterized that the compounds are dialkyl ethers. Method according to claim 7, characterized that the denaturing reagent is diethylene glycol dimethyl ether, DME, act. Method according to at least one of claims 5 to 8, characterized in that the denaturing reagent in concentrations between 5 and 25%. Method according to at least one of claims 1 to 9, characterized in that the concentration of bisulfite 1 to 6 mol / l. Method according to at least one of claims 1 to 10, characterized in that the reaction base temperature is 30-70 ° C. Method according to claim 11, characterized in that the basic reaction temperature is 45-60 ° C. Method according to at least one of claims 1 to 12, characterized in that the number of short-term temperature increases, Thermospikes, 1 to 10. Method according to claim 13, characterized in that that the number of short-term temperature increases, thermospikes, 2 to 5 is. Method according to at least one of claims 1 to 14, characterized in that the short-term temperature increases, Thermospikes, raise the reaction temperature to 85 to 100 ° C. Method according to claim 15, characterized in that that the short-term temperature increases, thermospikes, the reaction temperature at 90 to 98 ° C increase. Method according to at least one of claims 1 to 16, characterized in that the purification of the converted DNA over Ultrafiltration takes place. Method according to at least one of claims 5 to 8, characterized in that the purification of the converted DNA by means of magnetic particles. Method according to at least one of claims 1 to 18, characterized in that the converted DNA by means of a the following methods are analyzed: MSP, Heavy Methyl, MsSNuPE, methyl-light. Method according to at least one of claims 1 to 19, characterized in that DNA from tissue samples or body fluids is examined. Use of the method after at least one the claims 1 to 20 for methylation analysis, Use of the method after at least one the claims 1 to 20 for the diagnosis or prognosis of cancer or other with a change predicted disease associated with cytosine methylation status of unwanted Drug effects, establishing a specific drug therapy, for monitoring the success of a drug therapy, to distinguish Cell types or tissues and to study cell differentiation.