By a News Reporter-Staff News Editor at Investment Weekly News -- According to news reporting originating from Alexandria, Virginia, by VerticalNews journalists, a patent by the inventors Markert-Hahn, Christine (Penzberg, DE); Block, Dirk (Bichl, DE), filed on August 24, 2015, was published online on January 16, 2018.
The assignee for this patent, patent number 9868756, is Epigenomics Ag (Berlin, DE).
Reporters obtained the following quote from the background information supplied by the inventors: "Field of the Invention
"The present application is directed to a method for performing a bisulfite reaction to determine methylation positions in a nucleic acid, i.e. methylated and non-methylated cytosines, whereby the nucleic acid is bound to a solid phase during the deamination and/or desulfonation step of the bisulfite reaction. The solid phase is preferably a material comprising glass or silica, more preferably a glass fleece, glass membrane or a magnetic glass particle. Further, the use of a solid phase for binding a nucleic acid during the deamination and/or desulfonation step of the bisulfite reaction is disclosed and a kit containing a bisulfite reagent and a solid phase.
"Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding desoxyribonucleic acid (DNA) presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements requires effective mechanisms for its long term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. DNA methylation is essential for the development of mammals; and plays a potential role during aging and cancer. The involvement of methylation in the regulation of gene expression and as an epigenetic modification marking imprinted genes is well established. In mammals, methylation occurs only at cytosine residues and more specifically only on cytosine residues adjacent to a guanosine residue, i.e. at the sequence CG. The detection and mapping of DNA methylation sites are essential steps towards understanding the molecular signals which indicate whether a given sequence is methylated.
"Description of Related Art
"This is currently accomplished by the so-called bisulfite method described by Frommer, M., et al., Proc Natl Acad Sci USA 89 (1992) 1827-31) for the detection of 5-methyl-cytosines. The bisulfite method of mapping 5-methylcytosine uses the effect that sodium hydrogen sulfite reacts with cytosine but not or only poorly with 5-methyl-cytosine. Cytosine reacts with bisulfite to form a sulfonated cytosine reaction intermediate being prone to deamination resulting in a sulfonated uracil which can be desulfonated to uracil under alkaline conditions. It is common knowledge that uracil has the base pairing behavior of thymine different to the educt cytosine whereas 5-methylcytosine has the base pairing behavior of cytosine. This makes the discrimination of methylated or non-methylated cytosines possible by e.g. bisulfite genomic sequencing (Grigg, G. and Clark, S., Bioessays 16 (1994) 431-6; Grigg, G. W., DNA Seq 6 (1996) 189-98) or methylation specific PCR (MSP) disclosed in U.S. Pat. No. 5,786,146.
"There are various documents addressing specific aspects of the bisulfite reaction (Benyajati, C., et al., Nucleic Acids Res 8 (1980) 5649-67) make general investigations to the bisulfite modification of 5-methyl-deoxycytosine and deoxycytosine (Olek, A., et al., Nucleic Acids Res 24 (1996) 5064-6) disclose a method for bisulfite base sequencing whereby bisulfite treatment and subsequent PCR steps are performed on material embedded in agarose beads. In the bisulfite method as disclosed by Clark, S. J., et al., Nucleic Acids Res 22 (1994) 2990-7, the sample is desalted after deamination. Raizis, A. M., et al., Anal Biochem 226 (1995) 161-6 disclose a bisulfite method of 5-methylcytosine mapping that minimizes template degradation. They investigate the influence of pH, temperature and time of reaction. Similar investigations have been made by Grunau, C., et al., Nucleic Acids Res 29 (2001) E65-5 or Warnecke, P. M., et al., Methods 27 (2002) 101-7. Different additional components in the bisulfite mixture are disclosed by WO 01/98528 or by Paulin, R., et al., Nucleic Acids Res 26 (1998) 5009-10. An additional bisulfite step after bisulfite treatment and PCR is disclosed in WO 02/31186. Komiyama, M. and Oshima, S., Tetrahedron Letters 35 (1994) 8185-8188) investigate the catalysis of bisulfite-induced deamination of cytosine in oligodeoxyribonucleotides.
"Kits for performing bisulfite treatments are commercially available from Intergen, distributed by Serologicals Corporation, Norcross, Ga., USA, e.g. CpGenome.TM. DNA modification kit.
"A variation of the bisulfite genomic sequencing method is disclosed by Feil, R., et al., Nucleic Acids Res 22 (1994) 695-6, whereby the genomic DNA is bound to glass beads after deamination and washed. After elution the nucleic acid is desulfonated. It is known that nucleic acids can be isolated by the use of their binding behavior to glass surfaces, e.g. adsorption to silica gel or diatomic earths, adsorption to magnetic glass particles (MGPs) or organo silane particles under chaotropic conditions. Extraction using solid phases usually contains the steps of adding the solution with the nucleic acids to the solid phase under conditions allowing binding of the substance of interest to the solid phase, removal of the remainder of the solution from the solid phase bound nucleic acids and subsequent release of the nucleic acids from the solid phase into a liquid eluate (sometimes called elution). The result of the such process is usually a solution containing the substance of interest in dissolved state."
In addition to obtaining background information on this patent, VerticalNews editors also obtained the inventors' summary information for this patent: "All prior art methods for the bisulfite treatment have disadvantages. Therefore, the problem to be solved by the present invention was to provide a method which overcomes the disadvantages of the prior art methods.
"The problem discussed above is solved by providing a method for the conversion of cytosine bases in a nucleic acid to uracil bases whereby 5-methyl-cytosine bases are not significantly converted ('bisulfite reaction' or 'bisulfite treatment') whereby the nucleic acid is bound to a solid phase during the deamination and/or desulfonation step of the bisulfite reaction. Preferably, the solid phase is a glass fleece, a glass membrane or a magnetic glass particle. Further, the present invention discloses uses of a solid phase in the deamination and for desulfonation step of the bisulfite reaction and kits containing a solid phase and reagents for performing a bisulfite reaction.
"The use of a solid phase during the deamination and/or desulfonation step of the bisulfite reaction, preferably in the desulfonation step, has the advantage that the handling is simpler and/or easily amenable to automation. For example, when glass fleeces are used for the deamination and/or desulfonation steps, no time consuming DNA precipitation reactions are necessary; bound-free separation can easily be achieved by centrifugation, the dead volume of glass fleece is neglectable and therefore washing steps are very effective. This is an advantage when the bisulfite treatment DNA is used for PCR where potential inhibitors can reduce the sensitivity significantly. The method according to the invention can be easily performed manually and is therefore well suited for smaller laboratories where routine analysers are not available. For larger laboratories with higher sample throughput, the use a solid phase that can be handled by routine analyzers, in particular magnetic glass particles, is advantageous. In a routine bisulfite reaction denaturing conditions are chosen as bisulfite can only react with pyrimidines that are not involved in base-pairing. Therefore, it is surprising that the bisulfite reaction can be performed successfully in a satisfying manner by the method according to the invention as the nucleic acid is bound to the surface of the solid phase by various interactions possibly influencing the success of the bisulfite reaction."
For more information, see this patent: Markert-Hahn, Christine; Block, Dirk. Method for Bisulfite Treatment. U.S. Patent Number 9868756, filed August 24, 2015, and published online on January 16, 2018. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=9868756.PN.&OS=PN/9868756RS=PN/9868756
Keywords for this news article include: Europe, Germany, Business, Genetics, DNA Research, Epigenomics Ag.
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