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Method for the carry-over protection in dna amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acidsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidMethod for the carry-over protection in dna amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080096199, Method for the carry-over protection in dna amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] In recent decades in molecular biology studies have focused primarily on genes, the transcription of those genes into RNA, and the translation of the RNA into protein. There has been a more limited analysis of the regulatory mechanisms associated with gene control. Gene regulation, for example, at what stage of development of the individual a gene is activated or inhibited, and the tissue specific nature of this regulation is less understood. However, it can be correlated with a high degree of probability to the extent and nature of methylation of the gene or genome. Specific cell types can be correlated with specific methylation patterns and this has been shown for a number of cases (Adorjan et al. (2002) Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res. 30 (5) e21). [0002] In higher order eukaryotes DNA is methylated nearly exclusively at cytosines located 5' to guanine in the CpG dinucleotide. This modification has important regulatory effects on gene expression, especially when involving CpG rich areas, known as CpG islands, located in the promoter regions of many genes. While almost all gene-associated islands are protected from methylation on autosomal chromosomes, extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X-chromosome of females. [0003] The cytosine's modification in form of methylation contains significant information. It is obvious that the identification of 5-methylcytosine in a DNA sequence as opposed to unmethylated cytosine is of greatest importance to analyze its role further. But, because the 5-methylcytosine behaves just as a cytosine for what concerns its hybridization preference (a property relied on for sequence analysis) its position cannot be identified by a normal sequencing reaction. [0004] Furthermore, in any amplification, such as a PCR amplification, this relevant epigenetic information, methylated cytosine or unmethylated cytosine, will be completely lost. [0005] Several methods are known that solve this problem. Usually genomic DNA is treated with a chemical or enzyme leading to a conversion of the cytosine bases, which consequently allows to differentiate the bases afterwards. The most common methods are a) the use of methylation sensitive restriction enzymes capable of differentiating between methylated and unmethylated DNA and b) the treatment with bisulfite. The use of said enzymes is limited due to the selectivity of the restriction enzyme towards a specific recognition sequence. [0006] Therefore, the `bisulfite treatment`, allowing for the specific reaction of bisulfite with cytosine, which, upon subsequent alkaline hydrolysis, is converted to uracil, whereas 5-methylcytosine remains unmodified under these conditions (Shapiro et al. (1970) Nature 227: 1047) is currently the most frequently used method for analyzing DNA for 5-methylcytosine. Uracil corresponds to thymine in its base pairing behavior, that is it hybridizes to adenine; whereas 5-methylcytosine does not change its chemical properties under this treatment and therefore still has the base pairing behavior of a cytosine, that is hybridizing with guanine. Consequently, the original DNA is converted in such a manner that 5-methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using "normal" molecular biological techniques, for example, amplification and hybridization or sequencing. All of these techniques are based on base pairing, which can now be fully exploited. Comparing the sequences of the DNA with and without bisulfite treatment allows an easy identification of those cytosines that have been unmethylated. [0007] An overview of the further known methods of detecting 5-methylcytosine may be gathered from the following review article: Fraga F M, Esteller M, Biotechniques 2002 September; 33(3):632, 634, 636-49. [0008] As the use of methylation-specific enzymes is restricted to certain sequences (comprising restriction sites), most methods are based on a bisulfite treatment that is conducted before a detection or amplifying step (for review: DE 100 29 915, A1 p. 2, lines 35-46 or the according translated U.S. application Ser. No. 10/311,661, see also WO 2004/067545). The term `bisulfite treatment` is meant to comprise treatment with a bisulfite, a disulfite or a hydrogensulfite solution. As known to the expert skilled in the art and according to the invention, the term "bisulfite" is used interchangeably for "hydrogensulfite". [0009] Several protocols are known in the art. However, all of the described protocols, comprise of the following steps: The genomic DNA is isolated, denatured, converted several hours by a concentrated bisulfite solution and finally desulfonated and desalted (e.g.: Frommer et al.: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA. 1992 Mar. 1; 89(5):1827-31). [0010] In recent times several technical improvements of the bisulfite methods were developed. [0011] The agarose bead method incorporates the DNA to be investigated in an agarose matrix, through which diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek A. et al. A modified and improved method for bisulphite based cytosine methylation analysis, Nucl. Acids Res. 1996, 24, 5064-5066). [0012] In the patent application WO 01/98528 (20040152080) a bisulfite conversion is described in which the DNA sample is incubated with a bisulfite solution of a concentration range between 0.1 mol/l to 6 mol/l in presence of a denaturing reagent and/or solvent and at least one scavenger. In said patent application several suitable denaturing reagents and scavengers are described. The final step is incubation of the solution under alkaline conditions whereby the deaminated nucleic acid is desulfonated. [0013] In the patent application WO 03/038121 (US 20040115663) a method is disclosed in which the DNA to be analysed is bound to a solid surface during the bisulfite treatment. Consequently, purification and washing steps are facilitated. [0014] In the patent application WO 04/067545 a method is disclosed in which the DNA sample is denatured by heat and incubated with a bisulfite solution of a concentration range between 3 mol/l to 6.25 mol/l. Thereby the pH value is between 5.0 and 6.0 and the nucleic acid is deaminated. Finally an incubation of the solution under alkaline conditions takes place, whereby the deaminated nucleic acid is desulfonated. [0015] The understanding in the art that a "bisulfite conversion" usually comprises the step of desulfonation can for example be taken from said application: "According to the invention the term a "bisulfite reaction", "bisulfite treatment" or "bisulfite method" shall mean a reaction for the conversion of a cytosine base, preferably cytosine bases, in a nucleic acid to an uracil base, preferably uracil bases, in the presence of bisulfite ions whereby preferably a 5-methyl-cytosine base, preferably 5-methyl-cytosine bases, is not significantly converted. This reaction for the detection of methylated cytosines is described in detail by Frommer et al., supra and Grigg and Clark (Grigg, G. and Clark, S., Bioessays 16 (1994) 431-436). The bisulfite reaction contains a deamination step and a desulfonation step, which can be conducted separately or simultaneously (see FIG. 1; Grigg and Clark, supra). The statement that 5-methyl-cytosine bases are not significantly converted shall only take the fact into account that it cannot be excluded that a small percentage of 5-methyl-cytosine bases is converted to uracil although it is intended to convert only and exclusively the (non-methylated) cytosine bases (Frommer et al., supra). The expert skilled in the art knows how to perform the bisulfite reaction, e.g. by referring to Frommer et al., supra or Grigg and Clark, supra who disclose the principal parameters of the bisulfite reaction." [0016] Furthermore in said application it is described what the general state of the art is with regard to the different protocols: "From Grunau et al., supra, it is known to the expert in the field what variations of the bisulfite method are possible. In summary, in the deamination step a buffer containing bisulfite ions, optionally chaotropic agents and optionally further reagents as an alcohol or stabilizers as hydroquinone are employed and the pH is in the acidic range. The concentration of bisulfite is between 0.1 and 6 M bisulfite, preferably between 1 M and 5.5 M, the concentration of the chaotropic agent is between 1 and 8 M, whereby preferably guanidinium salts are employed, the pH is in the acidic range, preferably between 4.5 and 6.5, the temperature is between 0.degree. C. and 90.degree. C., preferably between room temperature (25.degree. C.) and 90.degree. C., and the reaction time is between 30 min and 24 hours or 48 hours or even longer, but preferably between 1 hour and 24 hours. The desulfonation step is performed by adding an alkaline solution or buffer as e. g. a solution only containing a hydroxide, e. g sodium hydroxide, or a solution containing ethanol, sodium chloride and sodium hydroxide (e. g. 38% EtOH, 100 mM NaCI, 200 mM NaOH) and incubating at room temperature or elevated temperatures for several min, preferably between 5 min and 60 min." [0017] It is therefore clear that the desulfonation is an inherent feature of all of these methods, in any case a desulfonation takes place before the nucleic acids are used as templates for amplification reactions, in order to provide an ideal template for the polymerase utilized in the following reactions. [0018] In the patent application WO 05/038051 improvements for the conversion of unmethylated cytosine to uracil by treatment with a bisulfite reagent are described. According to this method the reaction is carried out in the presence of 10-35% by volume, preferentially in the presence of 20-30% by volume of dioxane, one of its derivatives or a similar aliphatic cyclic ether. The bisulfite reaction can also be carried out in the presence of a n-alkylene glycol compound, particularly in the presence of their dialkyl ethers, and especially in the presence of diethylene glycol dimethyl ether (DME). These compounds can be present in a concentration of 1-35 % by volume, preferentially of 5-25% by volume. According to this invention the bisulfite conversion is conducted at a temperature in the range of 0-80.degree. C. and that the reaction temperature is increased for 2 to 5 times to a range of 85-100.degree. C. briefly during the course of the conversion (thermospike). It is further preferred that the temperature increases to 85-100.degree. C., in particular to 90-98.degree. C. during the temperature increase of brief duration. [0019] Subsequent to a bisulfite treatment, usually short, specific fragments of a known gene are amplified and either completely sequenced (Olek A, Walter J. (1997) The pre-implantation ontogeny of the H19 methylation imprint. Nat. Genet. 3: 275-6) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L and Jones P A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25 :2529-31, WO 95/00669) or by enzymatic digestion (Xiong Z, Laird P W. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25: 2535-4). [0020] Another technique to detect hypermethylation is the so-called methylation specific PCR (MSP) (Herman J G, Graff J R, Myohanen S, Nelkin B D and Baylin S B. (1996), Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 93: 9821-6). The technique is based on the use of primers that differentiate between a methylated and a non-methylated sequence if applied after bisulfite treatment of said DNA sequence. The primer either contains a guanine at the position corresponding to the cytosine in which case it will after bisulfite treatment only bind if the position was methylated. Or the primer contains an adenine at the corresponding cytosine position and therefore only binds to said DNA sequence after bisulfite treatment if the cytosine was unmethylated and has hence been altered by the bisulfite treatment so that it hybridizes to adenine. With the use of these primers, amplicons can be produced specifically depending on the methylation status of a certain cytosine and will as such indicate its methylation state. [0021] Another technique is the detection of methylation via a labelled probe, such as used in the so called Tagman PCR, also known as MethyLight (U.S. Pat. No. 6,331,393). With this technique it became feasible to determine the methylation state of single or of several positions directly during PCR, without having to analyze the PCR products in an additional step. [0022] In addition, detection by hybridization has also been described (Olek et al., WO 99/28498). [0023] The treatment with bisulfite (or similar chemical agents or enzymes) with the effect of altering the base pairing behaviour of one type of cytosine specifically, either the methylated or the unmethylated, thereby introducing different hybridisation properties, makes the treated DNA more applicable to the conventional methods of molecular biology, especially the polymerase based amplification methods, such as the PCR. [0024] Base excision repair occurs in vivo to repair DNA base damage involving relatively minor disturbances in the helical DNA structure, such as deaminated, oxidized, alkylated or absent bases. Numerous DNA glycosylases are known in the art, and function in vivo during base excision repair to release damaged or modified bases by cleavage of the glycosidic bond that links such bases to the sugar phosphate backbone of DNA (Memisoglu, Samson, Mutation Res. (2000), 451:39-51). All DNA glycosylases cleave glycosidic bonds but differ in their base substrate specificity and in their reaction mechanisms. Continue reading about Method for the carry-over protection in dna amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids... 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