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    Method for the detection of cytosine methylations in DNA
    1.
    发明授权
    Method for the detection of cytosine methylations in DNA 有权

    公开(公告)号：US09863001B2

    公开(公告)日：2018-01-09

    申请号：US10568300

    申请日：2004-08-13

    申请人： Reimo Tetzner ,  Jürgen Distler

    发明人： Reimo Tetzner ,  Jürgen Distler

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6886 ,  C12Q2600/106 ,  C12Q2600/154

    摘要： The invention relates to a method for analyzing cytosine methylations in DNA sequences, according to which non-methylated cytosines are first converted into uracil while 5-methylcytosine remains unmodified. The DNA is then amplified by means of a polymerase and at least one primer whose 5 end is connected to a probe via a linker. The probe is intramolecularly hybridized onto the amplified products in accordance with the methylation state of the DNA, hybridization being detectable via different detection systems. The inventive method is particularly suitable for diagnosing and predicting cancer diseases and other diseases associated with a modification of the methylation state as well as for predicting undesired effects of medicaments.

    Method for the Quantification of Methylated DNA
    3.
    发明申请
    Method for the Quantification of Methylated DNA 有权
    甲基化DNA的定量方法

    公开(公告)号：US20110104663A1

    公开(公告)日：2011-05-05

    申请号：US11989190

    申请日：2006-07-20

    申请人： Reimo Tetzner

    发明人： Reimo Tetzner

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6827 ,  C12Q1/6809 ,  C12Q1/6818 ,  C12Q2561/113 ,  C12Q2535/131 ,  C12Q2523/125

    摘要： The method according to the invention concerns in particular a method for the quantification of methylated DNA. For this purpose, the DNA to be examined is first transformed such that unmethylated cytosine is converted to uracil while 5-methylcytosine remains unchanged. Subsequently, the transformed DNA is amplified in the presence of a pair of real-time probes. For this, a probe is constructed, which is specific for the methylated or for the unmethylated state of the DNA, and a probe, which binds methylation-unspecifically to the amplificate. The ratio of the signal intensities of the probes or the CT values allows for the calculation of the degree of methylation of the examined DNA. The method according to the invention is suited particularly for the diagnosis and prognosis of cancer and other diseases associated with a change in the methylation status, as well as, prediction of adverse for side-effects of pharmaceuticals.

    摘要翻译： 根据本发明的方法特别涉及用于定量甲基化DNA的方法。 为此目的，首先将待检测的DNA转化成未甲基化胞嘧啶转化为尿嘧啶，而5-甲基胞嘧啶保持不变。 随后，在一对实时探针的存在下扩增转化的DNA。 为此，构建了针对DNA的甲基化或未甲基化状态特异的探针，以及探针，其将甲基化特异性结合于扩增子。 探针的信号强度或CT值的比值允许计算被检DNA的甲基化程度。 根据本发明的方法特别适用于与甲基化状态改变相关的癌症和其它疾病的诊断和预后，以及对药物副作用的不良预测。

    Heavymethyl assay for the methylation analysis of the gstpi gene
    5.
    发明申请
    Heavymethyl assay for the methylation analysis of the gstpi gene 审中-公开
    gstpi基因甲基化分析的重甲基检测

    公开(公告)号：US20070117093A1

    公开(公告)日：2007-05-24

    申请号：US10562196

    申请日：2004-06-24

    申请人： Reimo Tetzner ,  Jurgen Distler

    发明人： Reimo Tetzner ,  Jurgen Distler

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6886 ,  C12Q1/6827 ,  C12Q2600/154 ,  C12Q2523/125

    摘要： Described herein is a method for the detection of cyto-sine methylation in DNA samples, wherein the following steps are conducted: a genomic DNA sample, which comprises the DNA to be investigated as well as background DNA is treated with bisulfite (= disulfite, hydrogen sulfite) in such a way that all of the unmethylated cytosine bases are converted to uracil, while the 5-methylcytosine bases remain unchanged; the bisulfite treated DNA sample is amplified with the use of at least 2 primer oligonucleotides as well as a polymerase, wherein the DNA to be investigated is pre-ferred over the background DNA as the template, and a control fragment is amplified simultaneously to the am-plification of the bisulfite treated DNA within the same reaction mixture the amplified products are analyzed and the methylation status in the DNA to be investigated is concluded from the presence and /or the amount of the amplified products and/or from the analysis of additional positions.

    摘要翻译： 本文描述了用于检测DNA样品中细胞正辛基甲基化的方法，其中进行以下步骤：将包含待研究的DNA以及背景DNA的基因组DNA样品用亚硫酸氢盐（=二亚硫酸盐，氢气 亚硫酸盐），使得所有未甲基化的胞嘧啶碱基转化成尿嘧啶，而5-甲基胞嘧啶碱基保持不变; 使用至少2个引物寡核苷酸和聚合酶扩增亚硫酸氢盐处理的DNA样品，其中待研究的DNA优先于背景DNA作为模板，并将对照片段同时扩增至上述 在相同的反应混合物中分析亚硫酸氢盐处理的DNA，分析扩增产物，并从待测量的DNA中的甲基化状态从扩增产物的存在和/或量得出和/或从额外位置的分析得出 。

    Method for the carry-over protection in DNA amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids
    9.
    发明授权
    Method for the carry-over protection in DNA amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids 有权
    用于DNA扩增系统中的遗传保护的方法，其靶向通过核酸的修饰预处理实现的甲基化分析

    公开(公告)号：US07700282B2

    公开(公告)日：2010-04-20

    申请号：US11248721

    申请日：2005-10-11

    申请人： Reimo Tetzner ,  Dimo Dietrich

    发明人： Reimo Tetzner ,  Dimo Dietrich

    IPC分类号： C12Q1/70 ,  C12P19/34

    CPC分类号： C12Q1/6876 ,  C12P19/34 ,  C12Q1/6806 ,  C12Q1/6827 ,  C12Q1/6848 ,  C12Q1/6883 ,  C12Q1/6886 ,  C12Q2600/156 ,  C12Q2523/125 ,  C12Q2521/531 ,  C12Q2531/113

    摘要： Particular aspects provide methods for specific amplification of template DNA in the presence of potentially contaminating PCR products from previous amplification experiments. Particular embodiments comprise, in a first step, contacting DNA with a bisulfite solution, which sulfonates unmethylated (but not methylated) cytosines, resulting in cytosine deamination and generation of sulfonated uracil. Such sulfonation protects the template nucleic acid from being a target for the enzyme uracil-DNA-glycosylase (UNG), whereas any contaminating DNA, which contains unprotected unsulfonated or desulfonated uracils, is degraded enzymatically while the UNG is active. After UNG treatment and inactivation thereof, the sulfonated uracil bases are converted into uracil by desulfonation. Such aspects have substantial utility for decontamination of nucleic acid samples; e.g., for avoiding amplification of ‘carry over products’ in the context of DNA methylation analysis. In further aspects, the inventive methods can be generally used as simplified methods of bisulfite treatment.

    摘要翻译： 在以前的扩增实验中存在可能污染的PCR产物的情况下，特定方面提供用于特异性扩增模板DNA的方法。 具体实施方案包括在第一步中使DNA与亚硫酸氢盐溶液接触，所述亚硫酸氢盐溶液磺化非甲基化（但不是甲基化的）胞嘧啶，导致胞嘧啶脱氨基并产生磺化的尿嘧啶。 这种磺化保护模板核酸不是酶尿嘧啶-DNA-糖基化酶（UNG）的靶标，而任何含有未保护的未磺化或脱磺酸尿嘧啶的污染性DNA在UNG活性时被酶促降解。 在UNG处理和灭活后，通过脱磺酸将磺化的尿嘧啶碱基转化成尿嘧啶。 这些方面对于核酸样品的净化具有实质性的实用性; 例如，为了避免在DNA甲基化分析的上下文中“携带产物”的扩增。 在另外的方面，本发明的方法通常可以用作亚硫酸氢盐处理的简化方法。

    METHOD FOR METHYLATION ANALYSIS OF NUCLEIC ACID
    10.
    发明申请
    METHOD FOR METHYLATION ANALYSIS OF NUCLEIC ACID 有权
    核酸甲基化分析方法

    公开(公告)号：US20100092951A1

    公开(公告)日：2010-04-15

    申请号：US12310051

    申请日：2007-08-01

    申请人： Reimo Tetzner ,  Joern Lewin

    发明人： Reimo Tetzner ,  Joern Lewin

    IPC分类号： C12Q1/68 ,  G01N33/50

    CPC分类号： C12Q1/6858 ,  C12Q2537/113 ,  C12Q2535/119 ,  C12Q2523/125

    摘要： The present invention relates to a method for methylation analysis. It comprises the providing of a double stranded nucleic acid; its conversion, whereby unmethylated bases become distinguishable in their base-pairing behavior from methylated bases, and the analysis of both of the converted nucleic acid strands.

    摘要翻译： 本发明涉及甲基化分析方法。 它包括提供双链核酸; 其转化，其中未甲基化的碱基在其碱基配对行为与甲基化碱基之间变得可区分，以及两个转化的核酸链的分析。