公开(公告)号：US20070048757A1

公开(公告)日：2007-03-01

申请号：US11421460

申请日：2006-05-31

申请人： Kai Lao ,  Neil Straus ,  Will Bloch

发明人： Kai Lao ,  Neil Straus ,  Will Bloch

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6809 ,  C12Q1/6853 ,  C12Q2537/143 ,  C12Q2531/113 ,  C12Q2525/207 ,  C12Q2533/101 ,  C12Q2525/301

摘要： The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. In some embodiments, the present teachings provide methods of forming micro RNA signatures from single cells, including stem cells. In some embodiments, the present teachings provide methods for determining the identity and/or purity of cells. The present employ performing a multiplexed reverse transcription reaction comprising stem-loop reverse transcription primers, which optionally undergoes temperature cycling, followed by a multiplexed PCR-based pre-amplification reaction, and a subsequently a plurality of lower-plex decoding PCRs.

摘要翻译： 本教导提供用于逆转录和扩增小核酸如微RNA的方法，组合物和试剂盒。 在一些实施方案中，本教导提供从单细胞（包括干细胞）形成微RNA特征的方法。 在一些实施方案中，本教导提供了确定细胞的身份和/或纯度的方法。 本发明采用多重逆转录反应，其包括茎环逆转录引物，其可任选进行温度循环，随后进行基于多重PCR的预扩增反应，随后进行多个较低级解码PCR。

公开(公告)号：US08628925B2

公开(公告)日：2014-01-14

申请号：US13154128

申请日：2011-06-06

申请人： Will Bloch

发明人： Will Bloch

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12P19/34 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q2531/101 ,  C12Q2525/301 ,  C12Q2521/107 ,  C12Q2527/125

摘要： The present teachings provide novel methods for amplifying short nucleic acids. In some embodiments, the present teachings provide novel methods for linearly amplifying a collection of micro RNAs by using temperature cycling during a reverse transcription reaction. The cycling can comprise at least 20 cycles of an annealing temperature segment of 10° C.-30° C., and a denaturation temperature segment of 35° C.-60° C. In some embodiments, the temperature cycled reaction can comprise an osmolyte.

摘要翻译： 本教导提供了用于扩增短核酸的新方法。 在一些实施方案中，本发明提供了用于在逆转录反应期间通过使用温度循环线性扩增微RNA集合的新方法。 循环可以包括10℃-30℃的退火温度段和35℃-60℃的变性温度段的至少20个循环。在一些实施方案中，温度循环反应可以包括 渗透剂

公开(公告)号：US20050037361A1

公开(公告)日：2005-02-17

申请号：US10637466

申请日：2003-08-08

申请人： Sulekha Rao Coticone ,  Will Bloch

发明人： Sulekha Rao Coticone ,  Will Bloch

IPC分类号： G01N33/53 ,  C12N15/09 ,  C12Q1/68 ,  G01N33/566 ,  C12P19/34

CPC分类号： C12Q1/686 ,  C12Q1/6846 ,  C12Q1/6848 ,  C12Q1/6876 ,  C12Q2527/125 ,  C12Q2525/151

摘要： The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of 50% or less; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of betaine, sorbitol or mixtures thereof, effective to reduce stutter relative to the amount of stutter observed in the absence of betaine and/or sorbitol. The invention also provides compositions containing betaine and/or sorbitol, kits for amplifying microsatellites having a G+C content of 50% or less, and methods of using all of the foregoing.

公开(公告)号：US06841349B2

公开(公告)日：2005-01-11

申请号：US09850514

申请日：2001-05-07

申请人： Sulekha Rao Coticone ,  Will Bloch

发明人： Sulekha Rao Coticone ,  Will Bloch

IPC分类号： G01N33/53 ,  C12N15/09 ,  C12Q1/68 ,  G01N33/566 ,  C07H21/02 ,  C07H21/04

CPC分类号： C12Q1/686 ,  C12Q1/6846 ,  C12Q1/6848 ,  C12Q1/6876 ,  C12Q2527/125 ,  C12Q2525/151

摘要： The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of 50% or less; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of betaine, sorbitol or mixtures thereof, effective to reduce stutter relative to the amount of stutter observed in the absence of betaine and/or sorbitol. The invention also provides compositions containing betaine and/or sorbitol, kits for amplifying microsatellites having a G+C content of 50% or less, and methods of using all of the foregoing.

摘要翻译： 本发明提供了减少微卫星扩增中的口吃的方法，包括以下步骤：提供包含G + C含量为50％或更少的微卫星的样品; 使样品与至少一种具有核酸聚合酶活性的酶接触; 并将样品与酶温育足够的时间和足以扩增微卫星的条件; 其中在一定量的甜菜碱，山梨糖醇或其混合物的存在下进行培养，其有效地相对于在不存在甜菜碱和/或山梨醇的情况下观察到的口吃量减少口吃。 本发明还提供含有甜菜碱和/或山梨糖醇的组合物，用于扩增G + C含量为50％以下的微卫星的试剂盒，以及使用上述所有方法。

公开(公告)号：US5538871A

公开(公告)日：1996-07-23

申请号：US390256

申请日：1995-02-17

申请人： Gerard J. Nuovo ,  Will Bloch

发明人： Gerard J. Nuovo ,  Will Bloch

IPC分类号： A61B10/00 ,  B01L7/00 ,  C12M1/40 ,  C12N15/09 ,  C12Q1/68 ,  C12R1/19 ,  C12P19/34 ,  C07H21/04 ,  C12N15/00

CPC分类号： C12Q1/6841 ,  B01L7/52 ,  C12Q1/686

摘要： Improvements to the in situ polymerase chain reaction (PCR), a process of in vitro enzymatic amplification of specific nucleic acid sequences within the cells where they originate, can be achieved by changing the way that the enzymatic reaction is started. Reaction initiation is delayed until the start of PCR thermal cycling, either by withholding a subset of PCR reagents from the cellular preparation until the preparation has been heated to 50.degree. C. to 80.degree. C., immediately before thermal cycling is begun, or by adding to the PCR reagents a single-stranded DNA binding protein which blocks reaction at temperatures below about 50.degree. C. If the in situ PCR is performed on cellular preparations already attached to a microscope slide, thermal cycling also is facilitated by use of a thermal cycler sample block or compartment designed optimally to hold the microscope slide and any vapor barrier covering the slide.

摘要翻译： 原位聚合酶链反应（PCR）的改进，其可以通过改变酶反应开始的方式来实现其在其中起源的细胞内的特定核酸序列的体外酶促扩增过程。 反应开始被延迟直到PCR热循环的开始，通过从细胞制剂中扣留一组PCR试剂，直到制备在热循环开始之前立即加热至50℃至80℃，或通过 向PCR试剂中加入在低于约50℃的温度下阻断反应的单链DNA结合蛋白。如果在已经连接到显微镜载玻片上的细胞制剂上进行原位PCR，则通过使用热 循环仪样品块或隔室设计最佳，以保持显微镜载玻片和覆盖幻灯片的任何蒸汽屏障。

公开(公告)号：US20130011839A1

公开(公告)日：2013-01-10

申请号：US13611974

申请日：2012-09-12

申请人： Sulekha Rao Coticone ,  Will Bloch

发明人： Sulekha Rao Coticone ,  Will Bloch

IPC分类号： C12Q1/68 ,  C12N9/12 ,  C12P19/34

CPC分类号： C12Q1/686 ,  C12Q1/6846 ,  C12Q1/6848 ,  C12Q1/6876 ,  C12Q2527/125 ,  C12Q2525/151

摘要： The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of 50% or less; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of betaine, sorbitol or mixtures thereof, effective to reduce stutter relative to the amount of stutter observed in the absence of betaine and/or sorbitol. The invention also provides compositions containing betaine and/or sorbitol, kits for amplifying microsatellites having a G+C content of 50% or less, and methods of using all of the foregoing.

公开(公告)号：US20110143352A1

公开(公告)日：2011-06-16

申请号：US12769577

申请日：2010-04-28

申请人： Sulekha Rao COTICONE ,  Will Bloch

发明人： Sulekha Rao COTICONE ,  Will Bloch

IPC分类号： C12Q1/68 ,  C12N9/12

CPC分类号： C12Q1/686 ,  C12Q1/6846 ,  C12Q1/6848 ,  C12Q1/6876 ,  C12Q2527/125 ,  C12Q2525/151

摘要： The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of 50% or less; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of betaine, sorbitol or mixtures thereof, effective to reduce stutter relative to the amount of stutter observed in the absence of betaine and/or sorbitol. The invention also provides compositions containing betaine and/or sorbitol, kits for amplifying microsatellites having a G+C content of 50% or less, and methods of using all of the foregoing.

摘要翻译： 本发明提供了减少微卫星扩增中的口吃的方法，包括以下步骤：提供包含G + C含量为50％或更少的微卫星的样品; 使样品与至少一种具有核酸聚合酶活性的酶接触; 并将样品与酶温育足够的时间和足以扩增微卫星的条件; 其中在一定量的甜菜碱，山梨糖醇或其混合物的存在下进行培养，其有效地相对于在不存在甜菜碱和/或山梨醇的情况下观察到的口吃量减少口吃。 本发明还提供含有甜菜碱和/或山梨糖醇的组合物，用于扩增G + C含量为50％以下的微卫星的试剂盒，以及使用上述所有方法。

公开(公告)号：US5041378A

公开(公告)日：1991-08-20

申请号：US84479

申请日：1987-08-11

申请人： Robert J. Drummond ,  Will Bloch ,  Brian W. Matthews ,  Pamela L. Toy

发明人： Robert J. Drummond ,  Will Bloch ,  Brian W. Matthews ,  Pamela L. Toy

IPC分类号： C12N9/36 ,  C12N9/92 ,  C12P21/02

CPC分类号： C12N9/2462 ,  C12N9/92 ,  C12P21/02 ,  Y10S435/886

摘要： Xylose isomerase (XI) muteins useful in the conversion of glucose to fructose or xylose to xylulose are obtained in usable amounts by protein structural and recombinant DNA methods, including x-ray crystallography, cloning, computer graphic modeling and site-directed mutagenesis and expression of the bacterial DNA sequences encoding native procaryotic xylose isomerase. These native sequences are altered to encode the xylose isomerase muteins having improved catalytic function and/or thermostability.

摘要翻译： 通过蛋白质结构和重组DNA方法获得可用于将葡萄糖转化为果糖或木糖至木酮糖的木糖异构酶（XI）突变蛋白，包括x射线晶体学，克隆，计算机图形建模和定点诱变和表达 编码天然原核木糖异构酶的细菌DNA序列。 改变这些天然序列以编码具有改善的催化功能和/或热稳定性的木糖异构酶突变蛋白。

公开(公告)号：US4962189A

公开(公告)日：1990-10-09

申请号：US131646

申请日：1987-12-11

申请人： Will Bloch

发明人： Will Bloch

IPC分类号： A61K38/00 ,  C07K14/415 ,  C07K19/00

CPC分类号： C07K19/00 ,  C07K14/415 ,  A61K38/00 ,  Y10S514/885

摘要： The invention herein is directed to methods using Procion dyes to perform separations of interest in manipulating the NAD.sup.+ -independent ribotoxins. The methods are useful for preparing therapeutic agents containing these ribotoxins or their A polypeptide components. This separation method has been applied in particular to preparing hybrid toxins containing ricin toxins, both for purifying the resulting products and also for separating the components intended to be used in the preparation of these end products. In addition, a novel ricin isotoxin prepared using the method of the invention is disclosed.

摘要翻译： 本发明涉及使用Procion染料来操作感兴趣的分离物以操纵NAD + - 独立核毒素的方法。 该方法可用于制备含有这些核毒素或其A多肽组分的治疗剂。 该分离方法特别用于制备含有蓖麻毒素的杂合毒素，用于纯化所得产物，也用于分离用于制备这些最终产品的成分。 此外，公开了使用本发明的方法制备的新颖的蓖麻毒素异毒素。

公开(公告)号：US07556943B2

公开(公告)日：2009-07-07

申请号：US11421319

申请日：2006-05-31

申请人： Will Bloch

发明人： Will Bloch

IPC分类号： C12P19/34 ,  C12Q1/68

CPC分类号： C12P19/34 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q2531/101 ,  C12Q2525/301 ,  C12Q2521/107 ,  C12Q2527/125

摘要： The present teachings provide novel methods for amplifying short nucleic acids. In some embodiments, the present teachings provide novel methods for linearly amplifying a collection of micro RNAs by using temperature cycling during a reverse transcription reaction. The cycling can comprise at least 20 cycles of an annealing temperature segment of 10 C-30 C, and a denaturation temperature segment of 35 C-60 C. In some embodiments, the temperature cycled reaction can comprise an osmolyte.

摘要翻译： 本教导提供了用于扩增短核酸的新方法。 在一些实施方案中，本发明提供了用于在逆转录反应期间通过使用温度循环线性扩增微RNA集合的新方法。 循环可以包括至少20个循环的10℃-30℃的退火温度段和35℃-60℃的变性温度段。在一些实施方案中，温度循环反应可包含渗透剂。