公开(公告)号：US20050287553A1

公开(公告)日：2005-12-29

申请号：US11100779

申请日：2005-04-06

申请人： David Guetig ,  Dirk Habighorst ,  Antje Kluth ,  Armin Schmitt ,  Matthias Schuster ,  Ina Schwope

发明人： David Guetig ,  Dirk Habighorst ,  Antje Kluth ,  Armin Schmitt ,  Matthias Schuster ,  Ina Schwope

IPC分类号： C12Q1/68 ,  G06F19/00 ,  C12P19/34

CPC分类号： C12Q1/6823 ,  C12Q2565/101 ,  C12Q2535/131 ,  C12Q2545/114 ,  C12Q2523/125

摘要： Particular aspects of the present invention provide a method for quantification of two different variations of a DNA sequence. Particularly, the invention relates to a quantification of methylated DNA, and for this purpose, the test DNA is converted so that cytosine is converted to uracil, while 5-methylcytosine remains unchanged. The converted DNA is amplified by means of a real-time PCR, wherein two labeled real-time probe types are utilized: one specific for methylated DNA; and one for unmethylated DNA. Preferably, the degree of methylation of the test DNA is calculated from the ratio of the signal intensities of the probes or from the Ct values. The inventive methods have substantial utility for diagnosis and prognosis of cancer and other disorders associated with altered or characteristic DNA methylation status, as well as having substantial utility for analysis of SNPs, allelic expression, and prediction of drug response, drug interactions, among other uses.

摘要翻译： 本发明的特定方面提供了用于定量DNA序列的两个不同变体的方法。 特别地，本发明涉及甲基化DNA的定量，为此目的，转化测试DNA使得胞嘧啶转化为尿嘧啶，而5-甲基胞嘧啶保持不变。 通过实时PCR扩增转化的DNA，其中使用两种标记的实时探针类型：一种特异性用于甲基化DNA; 一个用于未甲基化的DNA。 优选地，根据探针的信号强度与Ct值的比值来计算测试DNA的甲基化程度。 本发明的方法具有用于诊断和预测与改变或特征性DNA甲基化状态相关的癌症和其他病症的实质性用途，并且具有用于分析SNP，等位基因表达和药物反应预测，药物相互作用以及其它用途的实质性用途 。

公开(公告)号：US20070254293A1

公开(公告)日：2007-11-01

申请号：US11660804

申请日：2005-08-09

申请人： David Guetig ,  Matthias Schuster ,  Juergen Distler

发明人： David Guetig ,  Matthias Schuster ,  Juergen Distler

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6853 ,  C12Q1/6858 ,  C12Q2525/186 ,  C12Q2523/125 ,  C12Q2565/301 ,  C12Q2535/125

摘要： The present invention relates to a method for the analysis of methylated cytosines in DNA. In the first step of the invention unmethylated cytosines in the DNA to be analysed are chemically converted into uracil while 5-methylcytosines remain unchanged. In a second step a methylation specific oligonucleotide carrying a non-extendable 3′ end is annealed to the converted DNA. Subsequently, the non-extendable 3′ terminus of the oligonucleotide is removed in case the oligonucleotide is bound to the DNA with the methylation status to be detected. Finally the unblocked oligonucleotide is extended, and the methylation status is concluded from the absence or presence of an extended oligonucleotide product. The method is preferably used for diagnosis and/or prognosis of adverse events for individuals, for distinguishing cell types and tissues, or for investigating cell differentiation.

摘要翻译： 本发明涉及DNA分析甲基化胞嘧啶的方法。 在本发明的第一步中，待分析DNA中的未甲基化胞嘧啶化学转化成尿嘧啶，而5-甲基胞嘧啶保持不变。 在第二步中，携带不可延伸的3'末端的甲基化特异性寡核苷酸与转化的DNA退火。 随后，在寡核苷酸与具有待检测甲基化状态的DNA结合的情况下，去除寡核苷酸的不可延伸的3'末端。 最后，未阻断的寡核苷酸被延长，甲基化状态从延伸的寡核苷酸产物的不存在或存在而得出。 该方法优选用于个体的不良事件的诊断和/或预后，用于区分细胞类型和组织，或用于调查细胞分化。

公开(公告)号：US09719131B2

公开(公告)日：2017-08-01

申请号：US12847005

申请日：2010-07-30

申请人： Alexander Olek ,  Christian Piepenbrock ,  Kurt Berlin ,  David Guetig

发明人： Alexander Olek ,  Christian Piepenbrock ,  Kurt Berlin ,  David Guetig

IPC分类号： C07H21/02 ,  C12Q1/68

CPC分类号： C12Q1/6827 ,  C12Q1/6883 ,  C12Q2600/154

摘要： A method is described for the detection of the degree of methylation of a specific cytosine in the sequence context 5′-CpG-3′ of a genomic DNA sample. In the first step, the genomic DNA is chemically treated in such a way that the cytosine bases are converted to uracil, but not the 5-methylcytosine bases. Then segments of the genomic DNA which contain the said specific cytosine are amplified, whereby the amplified products are given a detectable label and in the following steps the extent of hybridization of the amplified products on two classes of oligonucleotides is determined by detection of the label of the amplified products, and a conclusion is made on the extent of methylation of said specific cytosine in the genomic DNA sample from the ratio of the labels detected on the two classes of oligonucleotides as a consequence of the hybridization.

公开(公告)号：US20110136687A1

公开(公告)日：2011-06-09

申请号：US12847005

申请日：2010-07-30

申请人： Alexander Olek ,  Christian Piepenbrock ,  Kurt Berlin ,  David Guetig

发明人： Alexander Olek ,  Christian Piepenbrock ,  Kurt Berlin ,  David Guetig

IPC分类号： C40B30/04 ,  C12Q1/68 ,  C07H21/04 ,  C07H21/00 ,  C40B40/06 ,  C40B50/14

CPC分类号： C12Q1/6827 ,  C12Q1/6883 ,  C12Q2600/154

摘要： A method is described for the detection of the degree of methylation of a specific cytosine in the sequence context 5′-CpG-3′ of a genomic DNA sample. In the first step, the genomic DNA is chemically treated in such a way that the cytosine bases are converted to uracil, but not the 5-methylcytosine bases. Then segments of the genomic DNA which contain the said specific cytosine are amplified, whereby the amplified products are given a detectable label and in the following steps the extent of hybridization of the amplified products on two classes of oligonucleotides is determined by detection of the label of the amplified products, and a conclusion is made on the extent of methylation of said specific cytosine in the genomic DNA sample from the ratio of the labels detected on the two classes of oligonucleotides as a consequence of the hybridization.

摘要翻译： 描述了用于检测基因组DNA样品的序列上下文5'-CpG-3'中特定胞嘧啶的甲基化程度的方法。 在第一步中，以使胞嘧啶碱基转化成尿嘧啶而不是5-甲基胞嘧啶碱基的方式对基因组DNA进行化学处理。 然后扩增含有所述特异性胞嘧啶的基因组DNA片段，由此扩增产物被给予可检测标记，并且在随后的步骤中，扩增产物在两类寡核苷酸上的杂交程度通过检测 扩增产物，并且从基因组DNA样品中的两种类型的寡核苷酸上检测到的标记的比例作为杂交的结果，对所述特定胞嘧啶在基因组DNA样品中甲基化的程度进行了总结。