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1. 发明申请

US20160130643A1 ACCURATE IN VITRO COPYING OF DNA METHYLATION 审中-公开
标题翻译：精确的DNA甲基化复制
公开(公告)号：US20160130643A1
公开(公告)日：2016-05-12
申请号：US14929234
申请日：2015-10-30
申请人： Ite A. Laird-Offringa , Jinkeng Asong , Mihaela Campan , Po-Han Chen , Crystal N. Marconett , Ian Stuart Haworth
发明人： Ite A. Laird-Offringa , Jinkeng Asong , Mihaela Campan , Po-Han Chen , Crystal N. Marconett , Ian Stuart Haworth
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6848 , C12Q1/6858 , C12Q1/686 , C12Q2521/125 , C12Q2521/501 , C12Q2521/514 , C12Q2537/164
摘要： A method of copying a methylated nucleic acid molecule is provided. The method includes copying a nucleic acid molecule into a plurality of nucleic acid molecules; and contacting the plurality of nucleic acid molecules with a DNA methyltransferase enzyme and an E3 ubiquitin ligase. The method results in the copying of the methylated nucleic acid molecule.
摘要翻译：提供复制甲基化核酸分子的方法。该方法包括将核酸分子复制到多个核酸分子中; 并使多个核酸分子与DNA甲基转移酶和E3泛素连接酶接触。该方法导致甲基化核酸分子的复制。

2. 发明申请

US20080254474A1 DNA METHYLATION ANALYSIS BY DIGITAL BISULFITE GENOMIC SEQUENCING AND DIGITAL METHYLIGHT 有权
标题翻译：数位双螺旋基因测序和数字甲基化的DNA甲基化分析
公开(公告)号：US20080254474A1
公开(公告)日：2008-10-16
申请号：US12102783
申请日：2008-04-14
申请人： Peter W. Laird , Binh N. Trinh , Mihaela Campan , Daniel J. Weisenberger
发明人： Peter W. Laird , Binh N. Trinh , Mihaela Campan , Daniel J. Weisenberger
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q1/6851 , C12Q2537/143 , C12Q2523/125 , C12Q2563/159
摘要： Provided are novel sensitive methylation assays referred to herein as Digital MethyLight, comprising stochastically distributing and compartmentalizing bisulfite-treated genomic DNA over multiple PCR reaction wells for detection of individually methylated DNA molecules in a large background of unmethylated DNA. Digital Bisulfite Genomic DNA Sequencing methods are also provided for high-resolution DNA methylation information without subcloning. Background signal and PCR contaminants are diluted, while the ratio of primer to methylated template DNA is kept high. Preferably, biological fluid (e.g., urine, blood-based (e.g., plasma and/or serum)) samples are analyzed for cancer diagnosis, prognosis and surveillance. Multiplexed PCR formats may be implemented to enhance when using small DNA amounts. Compositions and methods for diagnosis and/or prognosis of breast cancer, comprising the use of FOXE1, CLDN5 and/or RUNX3 gene markers are also provided (SEQ ID NOS: 17, 16 and 18, respectively for respective CpG island sequences), and in preferred embodiments plasma or serum samples are used.
摘要翻译：提供了本文中称为数字MethyLight的新型敏感甲基化测定法，其包括在多个PCR反应孔上随机分布和分隔亚硫酸氢盐处理的基因组DNA，用于在非甲基化DNA的大背景下检测单独的甲基化DNA分子。还提供数字亚硫酸氢盐基因组DNA测序方法，用于高分辨率DNA甲基化信息，无亚克隆。背景信号和PCR污染物被稀释，而引物与甲基化模板DNA的比例保持较高。优选地，分析生物流体（例如，尿液，血液（例如血浆和/或血清））样品用于癌症诊断，预后和监测。可以实施多重PCR格式以在使用小的DNA量时增强。还提供了包括使用FOXE1，CLDN5和/或RUNX3基因标记物的乳腺癌诊断和/或预后的组合物和方法（分别针对各个CpG岛序列的SEQ ID NO：17,16和18），并且优选实施方案使用血浆或血清样品。

3. 发明授权

US09797005B2 High throughput method of DNA methylation haplotyping 有权
公开(公告)号：US09797005B2
公开(公告)日：2017-10-24
申请号：US12094952
申请日：2006-11-22
申请人： Mihaela Campan , Peter W. Laird , Allen S. Yang , Hui-Lee Wong
发明人： Mihaela Campan , Peter W. Laird , Allen S. Yang , Hui-Lee Wong
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6858 , C12Q2565/301 , C12Q2545/114 , C12Q2523/125
摘要： Particular aspects provide novel, high-throughput methods to quantify DNA methylation (e.g., at a single-base resolution) in an allele-specific manner. The methods comprise use of an allele-specific sequence polymorphism (e.g., allele-specific single nucleotide polymorphism; SNP) in sufficient proximity to a CpG methylation site to provide for distinguishing the methylation levels between two alleles. In particular aspects, after bisulfite modification, the genomic DNA region is PCR-amplified, and the product subjected to allele-specific pyrosequencing, and the percentage of methylation determined based on the percentage of cytosine to thymidine conversion. In further embodiments, MethyLight™ is used after bisulfite treatment. The inventive methodology has, for example, substantial utility for affording quantitative analyses in the regulation of analyses of X-inactivation, the allele-specific expression of genes (e.g., in the immune system) and junk DNA, etc., and in classifying an individual as to whether they have loss of imprinting (LOI).

4. 发明授权

US08642263B2 High throughput methods comprising analysis of repetitive element DNA methylation 有权
标题翻译：高通量方法包括重复元件DNA甲基化的分析
公开(公告)号：US08642263B2
公开(公告)日：2014-02-04
申请号：US11719033
申请日：2005-11-10
申请人： Peter W. Laird , Daniel J. Weisenberger , Mihaela Campan , Tifany I. Long
发明人： Peter W. Laird , Daniel J. Weisenberger , Mihaela Campan , Tifany I. Long
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6827 , C12Q2561/113 , C12Q2545/114 , C12Q2531/113
摘要： Preferred aspects provide novel high-throughput, sensitive methods (e.g., real-time PCR-based (MethyLight™) reactions) for detection and/or measurement of global genomic 5-methylcytosine content, based on measurement of DNA methylation of Alu, LINE-1 repetitive sequences, and the chromosome 1 centromeric satellite alpha and juxtacentromeric satellite 2 repeat sequences. Additional aspects provide sensitive methods for determining the amount of a DNA (e.g., in formalin-fixed, paraffin-embedded tissues). Combined (mean) use of Alu and Sat2 repeat methylation measurements provides for a surprisingly close correlation with global genomic 5-methylcytosine content measurements obtained by HPLC. Methylation of Alu repeats was determined to be closely associated with HPLC-based global methylation levels, as was methylation of satellite 2 and LINE-1 global genomic 5-methylcytosine content. The assays provide surrogate markers for global genomic 5-methylcytosine content analyzes, and have substantial utility for high-throughput and population-based studies (e.g., genetic and dietary influences on global DNA methylation, folate deficiency, MTHFR gene polymorphisms, etc).
摘要翻译：优选的方面提供新的高通量，敏感的方法（例如，基于实时PCR的（MethyLight TM）反应），用于检测和/或测量全局基因组5-甲基胞嘧啶含量，基于测量Alu的DNA甲基化， LINE-1重复序列，以及1号染色体着丝粒卫星α和juxtacentromeric卫星2重复序列。另外的方面提供用于确定DNA量的敏感方法（例如，在福尔马林固定的石蜡包埋的组织中）。 Alu和Sat2重复甲基化测量的组合（平均）使用提供了通过HPLC获得的全球基因组5-甲基胞嘧啶含量测量值的令人惊讶的密切相关性。 Alu重复的甲基化被确定为与基于HPLC的全球甲基化水平密切相关，卫星2和LINE-1全局基因组5-甲基胞嘧啶含量的甲基化也是如此。该测定法为全球基因组5-甲基胞嘧啶含量分析提供替代标记，并且对于高通量和基于人群的研究（例如，对全球DNA甲基化，叶酸缺乏，MTHFR基因多态性等的遗传和饮食影响）具有实用性。

5. 发明申请

US20120219946A1 DNA METHYLATION MARKERS ASSOCIATED WITH THE CPG ISLAND METHYLATOR PHENOTYPE (CIMP) IN HUMAN COLORECTAL CANCER 审中-公开
标题翻译：与人类癌症中的CPG岛状甲基化合物（CIMP）相关的DNA甲基化标记
公开(公告)号：US20120219946A1
公开(公告)日：2012-08-30
申请号：US13366192
申请日：2012-02-03
申请人： Peter W. Laird , Kimberly D. Siegmund , Mihaela Campan , Daniel J. Weisenberger , Tiffany I. Long
发明人： Peter W. Laird , Kimberly D. Siegmund , Mihaela Campan , Daniel J. Weisenberger , Tiffany I. Long
IPC分类号： C12Q1/68 , C07H21/04 , G01N33/574
CPC分类号： C12Q1/6886 , C12Q2600/112 , C12Q2600/154 , C12Q2600/156 , C12Q2600/158
摘要： Particular aspects confirm the existence of a CpG island methylator phenotype (CIMP) in colorectal cancer, and provide novel validated DNA methylation markers associated with CIMP. Additional aspects provide novel methods and compositions for: determining CIMP status in colorectal cancers, determining the relationship between CIMP status and other molecular features of the cancers (e.g., BRAF mutation, KRAS mutation and MSI status); determining the relationship between CIMP status and other variables (e.g., age, sex, tumor location, family history, race, country of origin, tumor characteristics (including, tumor type, tumor grade, invasive margin characteristics, lymphocyte infiltration characteristics, direct spread, lymph node spread, venous spread and type of residual adjacent polyp, if present)); and determining, between subgroups defined by CIMP status and BRAF mutations, effects of selected risk factors (e.g., body mass index, smoking history, alcohol intake, dietary folate intake, folate metabolic enzyme polymorphisms and history of hormonal use).
摘要翻译：特异性方面证实了结肠直肠癌中CpG岛甲基化表型（CIMP）的存在，并提供与CIMP相关的新的有效DNA甲基化标记。另外的方面提供了新的方法和组合物：确定结肠直肠癌中的CIMP状态，确定CIMP状态与癌症的其它分子特征（例如BRAF突变，KRAS突变和MSI状态）之间的关系; 确定CIMP状态与其他变量（如年龄，性别，肿瘤位置，家族史，种族，起源国家，肿瘤特征（包括肿瘤类型，肿瘤等级，浸润性边缘特征，淋巴细胞浸润特征，直接传播，淋巴结扩散，静脉扩散和残留相邻息肉的类型，如果存在））; 并确定由CIMP状态和BRAF突变所定义的亚组之间，选择的风险因素（例如体重指数，吸烟史，酒精摄入，饮食叶酸摄入，叶酸代谢酶多态性和激素使用史）的影响。

6. 发明申请

US20090053706A1 DNA METHYLATION MARKERS ASSOCIATED WITH THE CPG ISLAND METHYLATOR PHENOTYPE (CIMP) IN HUMAN COLORECTAL CANCER 失效
标题翻译：与人类癌症中的CPG岛状甲基化合物（CIMP）相关的DNA甲基化标记
公开(公告)号：US20090053706A1
公开(公告)日：2009-02-26
申请号：US11913535
申请日：2006-05-02
申请人： Peter W. Laird , KImberly D. Siegmund , Mihaela Campan , Daniel J. Weisenberg , Tiffany I. Long
发明人： Peter W. Laird , KImberly D. Siegmund , Mihaela Campan , Daniel J. Weisenberg , Tiffany I. Long
IPC分类号： C12Q1/68 , C12Q1/02 , G01N33/53 , C07H21/00
CPC分类号： C12Q1/6886 , C12Q2600/112 , C12Q2600/154 , C12Q2600/156 , C12Q2600/158
摘要： Particular aspects confirm the existence of a CpG island methylator phenotype (CIMP) in colorectal cancer, and provide novel validated DNA methylation markers associated with CIMP. Additional aspects provide novel methods and compositions for: determining CIMP status in colorectal cancers, determining the relationship between CIMP status and other molecular features of the cancers (e.g., BRAF mutation, KRAS mutation and MSI status); determining the relationship between CIMP status and other variables (e.g., age, sex, tumor location, family history, race, country of origin, tumor characteristics (including, tumor type, tumor grade, invasive margin characteristics, lymphocyte infiltration characteristics, direct spread, lymph node spread, venous spread and type of residual adjacent polyp, if present)); and determining, between subgroups defined by CIMP status and BRAF mutations, effects of selected risk factors (e.g., body mass index, smoking history, alcohol intake, dietary folate intake, folate metabolic enzyme polymorphisms and history of hormonal use).
摘要翻译：特异性方面证实了结肠直肠癌中CpG岛甲基化表型（CIMP）的存在，并提供与CIMP相关的新的有效DNA甲基化标记。另外的方面提供了新的方法和组合物：确定结肠直肠癌中的CIMP状态，确定CIMP状态与癌症的其它分子特征（例如BRAF突变，KRAS突变和MSI状态）之间的关系; 确定CIMP状态与其他变量（如年龄，性别，肿瘤位置，家族史，种族，起源国家，肿瘤特征（包括肿瘤类型，肿瘤等级，浸润性边缘特征，淋巴细胞浸润特征，直接传播，淋巴结扩散，静脉扩散和残留相邻息肉的类型，如果存在））; 并确定由CIMP状态和BRAF突变所定义的亚组之间，选择的风险因素（例如体重指数，吸烟史，酒精摄入，饮食叶酸摄入，叶酸代谢酶多态性和激素使用史）的影响。

7. 发明授权

US09290803B2 DNA methylation analysis by digital bisulfite genomic sequencing and digital methylight 有权
标题翻译：数字亚硫酸氢盐基因组测序和数字甲基化的DNA甲基化分析
公开(公告)号：US09290803B2
公开(公告)日：2016-03-22
申请号：US12102783
申请日：2008-04-14
申请人： Peter W. Laird , Binh N. Trinh , Mihaela Campan , Daniel J. Weisenberger
发明人： Peter W. Laird , Binh N. Trinh , Mihaela Campan , Daniel J. Weisenberger
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q1/6851 , C12Q2537/143 , C12Q2523/125 , C12Q2563/159
摘要： Provided are novel sensitive methylation assays referred to herein as Digital MethyLight, comprising stochastically distributing and compartmentalizing bisulfite-treated genomic DNA over multiple PCR reaction wells for detection of individually methylated DNA molecules in a large background of unmethylated DNA. Digital Bisulfite Genomic DNA Sequencing methods are also provided for high-resolution DNA methylation information without subcloning. Background signal and PCR contaminants are diluted, while the ratio of primer to methylated template DNA is kept high. Preferably, biological fluid (e.g., urine, blood-based (e.g., plasma and/or serum)) samples are analyzed for cancer diagnosis, prognosis and surveillance. Multiplexed PCR formats may be implemented to enhance when using small DNA amounts. Compositions and methods for diagnosis and/or prognosis of breast cancer, comprising the use of FOXE1, CLDN5 and/or RUNX3 gene markers are also provided (SEQ ID NOS: 17, 16 and 18, respectively for respective CpG island sequences), and in preferred embodiments plasma or serum samples are used.
摘要翻译：提供了本文称为数字MethyLight的新型敏感甲基化测定法，其包括在多个PCR反应孔上随机分布和分隔亚硫酸氢盐处理的基因组DNA，用于在非甲基化DNA的大背景下检测单独的甲基化DNA分子。还提供数字亚硫酸氢盐基因组DNA测序方法，用于高分辨率DNA甲基化信息，无亚克隆。背景信号和PCR污染物被稀释，而引物与甲基化模板DNA的比例保持较高。优选地，分析生物流体（例如，尿液，血液（例如血浆和/或血清））样品用于癌症诊断，预后和监测。可以实施多重PCR格式以在使用小的DNA量时增强。还提供了包括使用FOXE1，CLDN5和/或RUNX3基因标记物的乳腺癌诊断和/或预后的组合物和方法（分别针对各个CpG岛序列的SEQ ID NO：17,16和18），并且优选实施方案使用血浆或血清样品。

8. 发明授权

US08110361B2 DNA methylation markers associated with the CpG island methylator phenotype (CIMP) in human colorectal cancer 失效
标题翻译：与人结肠直肠癌中CpG岛甲基化表型（CIMP）相关的DNA甲基化标记
公开(公告)号：US08110361B2
公开(公告)日：2012-02-07
申请号：US11913535
申请日：2006-05-02
申请人： Peter W. Laird , Kimberly D. Siegmund , Mihaela Campan , Daniel J. Weisenberger , Tiffany I. Long
发明人： Peter W. Laird , Kimberly D. Siegmund , Mihaela Campan , Daniel J. Weisenberger , Tiffany I. Long
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6886 , C12Q2600/112 , C12Q2600/154 , C12Q2600/156 , C12Q2600/158
摘要： Particular aspects confirm the existence of a CpG island methylator phenotype (CIMP) in colorectal cancer, and provide novel validated DNA methylation markers associated with CIMP. Additional aspects provide novel methods and compositions for: determining CIMP status in colorectal cancers, determining the relationship between CIMP status and other molecular features of the cancers (e.g., BRAF mutation, KRAS mutation and MSI status); determining the relationship between CIMP status and other variables (e.g., age, sex, tumor location, family history, race, country of origin, tumor characteristics (including, tumor type, tumor grade, invasive margin characteristics, lymphocyte infiltration characteristics, direct spread, lymph node spread, venous spread and type of residual adjacent polyp, if present)); and determining, between subgroups defined by CIMP status and BRAF mutations, effects of selected risk factors (e.g., body mass index, smoking history, alcohol intake, dietary folate intake, folate metabolic enzyme polymorphisms and history of hormonal use).
摘要翻译：特异性方面证实了结肠直肠癌中CpG岛甲基化表型（CIMP）的存在，并提供与CIMP相关的新的有效DNA甲基化标记。另外的方面提供了新的方法和组合物：确定结肠直肠癌中的CIMP状态，确定CIMP状态与癌症的其它分子特征（例如BRAF突变，KRAS突变和MSI状态）之间的关系; 确定CIMP状态与其他变量（如年龄，性别，肿瘤位置，家族史，种族，起源国家，肿瘤特征（包括肿瘤类型，肿瘤等级，浸润性边缘特征，淋巴细胞浸润特征，直接传播，淋巴结扩散，静脉扩散和残留相邻息肉的类型，如果存在））; 并确定由CIMP状态和BRAF突变所定义的亚组之间，选择的风险因素（例如体重指数，吸烟史，酒精摄入，饮食叶酸摄入，叶酸代谢酶多态性和激素使用史）的影响。

9. 发明申请

US20090123915A1 HIGH THROUGHPUT METHODS COMPRISING ANALYSIS OF REPETITIVE ELEMENT DNA METHYLATION 有权
标题翻译：包含重复元件DNA甲基化分析的高通量方法
公开(公告)号：US20090123915A1
公开(公告)日：2009-05-14
申请号：US11719033
申请日：2005-11-10
申请人： Peter W. Laird , Daniel J. Weisenberger , Mihaela Campan , Tiffany I. Long
发明人： Peter W. Laird , Daniel J. Weisenberger , Mihaela Campan , Tiffany I. Long
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6827 , C12Q2561/113 , C12Q2545/114 , C12Q2531/113
摘要： Preferred aspects provide novel high-throughput, sensitive methods (e.g., real-time PCR-based (MethyLight™) reactions) for detection and/or measurement of global genomic 5-methylcytosine content, based on measurement of DNA methylation of Alu, LINE-1 repetitive sequences, and the chromosome 1 centromeric satellite alpha and juxtacentromeric satellite 2 repeat sequences. Additional aspects provide sensitive methods for determining the amount of a DNA (e.g., in formalin-fixed, paraffin-embedded tissues). Combined (mean) use of Alu and Sat2 repeat methylation measurements provides for a surprisingly close correlation with global genomic 5-methylcytosine content measurements obtained by HPLC. Methylation of Alu repeats was determined to be closely associated with HPLC-based global methylation levels, as was methylation of satellite 2 and LINE-1 global genomic 5-methylcytosine content. The assays provide surrogate markers for global genomic 5-methylcytosine content analyses, and have substantial utility for high-throughput and population-based studies (e.g., genetic and dietary influences on global DNA methylation, folate deficiency, MTHFR gene polymorphisms, etc).
摘要翻译：优选的方面提供新的高通量，敏感的方法（例如，基于实时PCR的（MethyLight TM）反应），用于检测和/或测量全局基因组5-甲基胞嘧啶含量，基于测量Alu的DNA甲基化， LINE-1重复序列，以及1号染色体着丝粒卫星α和juxtacentromeric卫星2重复序列。另外的方面提供用于确定DNA量的敏感方法（例如，在福尔马林固定的石蜡包埋的组织中）。 Alu和Sat2重复甲基化测量的组合（平均）使用提供了通过HPLC获得的全球基因组5-甲基胞嘧啶含量测量值的令人惊讶的密切相关性。 Alu重复的甲基化被确定为与基于HPLC的全球甲基化水平密切相关，卫星2和LINE-1全局基因组5-甲基胞嘧啶含量的甲基化也是如此。该测定法为全球基因组5-甲基胞嘧啶含量分析提供替代标记，并且对于高通量和基于人群的研究（例如，对全球DNA甲基化，叶酸缺乏，MTHFR基因多态性等的遗传和饮食影响）具有实用性。

10. 发明申请

US20080286787A1 High Throughput Method of DNA Methylation Haplotyping 有权
标题翻译： DNA甲基化单倍体的高通量方法
公开(公告)号：US20080286787A1
公开(公告)日：2008-11-20
申请号：US12094952
申请日：2006-11-22
申请人： Mihaela Campan , Peter W. Laird , Allen S. Yang , Hui-Lee Wong
发明人： Mihaela Campan , Peter W. Laird , Allen S. Yang , Hui-Lee Wong
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6858 , C12Q2565/301 , C12Q2545/114 , C12Q2523/125
摘要： Particular aspects provide novel, high-throughput methods to quantify DNA methylation (e.g., at a single-base resolution) in an allele-specific manner. The methods comprise use of an allele-specific sequence polymorphism (e.g., allele-specific single nucleotide polymorphism; SNP) in sufficient proximity to a CpG methylation site to provide for distinguishing the methylation levels between two alleles. In particular aspects, after bisulfite modification, the genomic DNA region is PCR-amplified, and the product subjected to allele-specific pyrosequencing, and the percentage of methylation determined based on the percentage of cytosine to thymidine conversion. In further embodiments, MethyLight™ is used after bisulfite treatment. The inventive methodology has, for example, substantial utility for affording quantitative analyses in the regulation of analyses of X-inactivation, the allele-specific expression of genes (e.g., in the immune system) and junk DNA, etc., and in classifying an individual as to whether they have loss of imprinting (LOI).
摘要翻译：特定方面提供以等位基因特异性方式定量DNA甲基化（例如，单碱基分辨率）的新颖的高通量方法。所述方法包括使用足够接近CpG甲基化位点的等位基因特异性序列多态性（例如，等位基因特异性单核苷酸多态性; SNP）以提供区分两个等位基因之间的甲基化水平。在特定方面，在亚硫酸氢盐修饰后，基因组DNA区域被PCR扩增，并且产物进行等位基因特异性焦磷酸测序，并且甲基化百分比基于胞嘧啶对胸苷转化的百分比而确定。在另外的实施方案中，在亚硫酸氢盐处理后使用MethyLight TM。例如，本发明的方法具有用于在X灭活的分析，基因（例如免疫系统）等等基因特异性表达和垃圾DNA等的分析中的定量分析以及在分类个人关于他们是否损失了印记（LOI）。

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