公开(公告)号：US07074600B2

公开(公告)日：2006-07-11

申请号：US10272465

申请日：2002-10-15

申请人： Frank B. Dean ,  Roger S. Lasken ,  Linhua Fang ,  A. Fawad Faruqi ,  Osama A. Alsmadi ,  Mark D. Driscoll ,  Seiyu Hosono ,  Michele Wisniewski ,  Wanmin Song

发明人： Frank B. Dean ,  Roger S. Lasken ,  Linhua Fang ,  A. Fawad Faruqi ,  Osama A. Alsmadi ,  Mark D. Driscoll ,  Seiyu Hosono ,  Michele Wisniewski ,  Wanmin Song

IPC分类号： C12P19/34

CPC分类号： C12Q1/6844 ,  C12Q1/6806 ,  C12Q2527/125 ,  C12Q2527/119 ,  C12Q2537/143 ,  C12Q2531/119 ,  C12Q2525/125

摘要： Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be φ29 DNA polymerase.

摘要翻译： 公开了用于扩增感兴趣的核酸序列的组合物和方法。 所公开的方法通常涉及靶序列的复制，使得在复制期间，复制的链通过另一复制链的链置换复制而从靶序列位移。 在所公开的方法的一种形式中，目标样品不经历变性条件。 已经发现，靶核酸，例如基因组DNA不需要被变性以用于有效的多位置扩增。 所使用的引物可以是六聚体引物。 引物还可以各自含有至少一个修饰的核苷酸，使得引物是耐核酸酶的。 引物还可以各自包含至少一个修饰的核苷酸，使得引物的融解温度相对于没有修饰的核苷酸的相同序列的引物而改变。 DNA聚合酶可以是phi29 DNA聚合酶。

公开(公告)号：US07205129B1

公开(公告)日：2007-04-17

申请号：US09514113

申请日：2000-02-28

申请人： Frank B. Dean ,  A. Fawad Faruqi

发明人： Frank B. Dean ,  A. Fawad Faruqi

IPC分类号： C12P19/34 ,  C12Q1/68 ,  C07H21/04

CPC分类号： C12Q1/6844 ,  C12Q1/6853

摘要： Disclosed are compositions and methods useful for reducing the formation of artifacts during nucleic acid amplification reactions. The method uses special oligonucleotides, referred to herein as template-deficient oligonucleotides, that cannot serve as a template for nucleic acid synthesis over part of their length. This prevents the oligonucleotides from serving as effective templates in the formation of artifacts. The disclosed method involves using a template-deficient oligonucleotide as at least one of the oligonucleotides (preferably a primer) in a nucleic acid amplification reaction, where the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, preferably at or near the 5′ end of the template-deficient oligonucleotide. The disclosed method is useful for reducing artifacts in any nucleic acid amplification reaction involving oligonucleotides. In a preferred form of the method the nucleic acid amplification reaction does not involve thermal cycling. The disclosed method is effective at reducing non-cycle oligonucleotide-based artifacts. Also disclosed are kits useful for reducing artifacts in nucleic acid amplification reactions. The disclosed kits include a template-deficient oligonucleotide, wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, and a nucleic acid polymerase.

摘要翻译： 公开了用于在核酸扩增反应期间减少伪影形成的组合物和方法。 该方法使用特定的寡核苷酸，本文称为模板缺陷型寡核苷酸，其不能作为核苷酸合成部分长度的模板。 这可以防止寡核苷酸作为伪像形成的有效模板。 所公开的方法包括在核酸扩增反应中使用模板缺陷型寡核苷酸作为寡核苷酸（优选引物）中的至少一种，其中模板缺陷型寡核苷酸包含一个或多个模板缺陷型核苷酸，优选为或接近5 “模板缺陷寡核苷酸的末端。 所公开的方法可用于减少涉及寡核苷酸的任何核酸扩增反应中的伪像。 在该方法的优选形式中，核酸扩增反应不涉及热循环。 所公开的方法在减少基于非循环寡核苷酸的伪像方面是有效的。 还公开了可用于减少核酸扩增反应中的伪像的试剂盒。 所公开的试剂盒包括模板缺陷型寡核苷酸，其中模板缺陷型寡核苷酸包含一个或多个模板缺陷核苷酸和核酸聚合酶。

公开(公告)号：US06977148B2

公开(公告)日：2005-12-20

申请号：US09977868

申请日：2001-10-15

申请人： Frank B. Dean ,  Roger S. Lasken ,  Linhua Fang ,  A. Fawad Faruqi ,  Osama A. Alsmadi ,  Mark D. Driscoll ,  Seiyu Hosono

发明人： Frank B. Dean ,  Roger S. Lasken ,  Linhua Fang ,  A. Fawad Faruqi ,  Osama A. Alsmadi ,  Mark D. Driscoll ,  Seiyu Hosono

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6844 ,  C12Q2527/125

摘要： Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be φ29 DNA polymerase.

摘要翻译： 公开了用于扩增感兴趣的核酸序列的组合物和方法。 所公开的方法通常涉及靶序列的复制，使得在复制期间，复制的链通过另一复制链的链置换复制而从靶序列位移。 在所公开的方法的一种形式中，目标样品不经历变性条件。 已经发现，靶核酸，例如基因组DNA不需要被变性以用于有效的多位置扩增。 所使用的引物可以是六聚体引物。 引物还可以各自含有至少一个修饰的核苷酸，使得引物是耐核酸酶的。 引物还可以各自包含至少一个修饰的核苷酸，使得引物的融解温度相对于没有修饰的核苷酸的相同序列的引物而改变。 DNA聚合酶可以是phi29 DNA聚合酶。

公开(公告)号：US06368801B1

公开(公告)日：2002-04-09

申请号：US09547757

申请日：2000-04-12

申请人： A. Fawad Faruqi

发明人： A. Fawad Faruqi

IPC分类号： C12Q168

CPC分类号： C12Q1/6844 ,  C12Q2521/501

摘要： Disclosed are techniques for detection of nucleic acids, amplification of nucleic acids, or both, involving ligation by T4 RNA ligase of DNA strands hybridized to an RNA strand. These techniques are particularly useful for the detection of RNA sequences and for amplification of nucleic acids from, or dependent on, RNA sequences. It has been discovered that T4 RNA ligase can efficiently ligate DNA ends of nucleic acid strands hybridized to an RNA strand. In particular, this ligation is more efficient than the same ligation carried out with T4 DNA ligase. Thus, techniques involving ligation of DNA ends of nucleic acid strands hybridized to RNA can be performed more efficiently by using T4 RNA ligase. Many known ligation-based detection and amplification techniques are improved through the use of T4 RNA ligase acting on DNA strands or ends. Such techniques include ligase chain reaction (LCR), ligation combined with reverse transcription polymerase chain reaction (RT PCR), ligation-mediated polymerase chain reaction (LMPCR), polymerase chain reaction/ligation detection reaction (PCR/LDR), ligation-dependent polymerase chain reaction (LD-PCR), oligonucleotide ligation assay (OLA), ligation-during-amplification (LDA), ligation of padlock probes, open circle probes, and other circularizable probes, and iterative gap ligation (IGL).

摘要翻译： 公开了用于检测核酸，核酸扩增或两者的技术，涉及通过T4 RNA连接酶与RNA链杂交的DNA链的连接。 这些技术特别可用于RNA序列的检测和用于扩增来自或依赖于RNA序列的核酸。 已经发现，T4 RNA连接酶可以有效地连接与RNA链杂交的核酸链的DNA末端。 特别地，该连接比用T4 DNA连接酶进行的相同连接更有效。 因此，可以通过使用T4 RNA连接酶更有效地进行涉及与RNA杂交的核酸链的DNA末端连接的技术。 通过使用作用于DNA链或末端的T4 RNA连接酶，许多已知的基于连接的检测和扩增技术得到改善。 这种技术包括连接酶链反应（LCR），结合逆转录聚合酶链反应（RT PCR），连接介导的聚合酶链反应（LMPCR），聚合酶链反应/连接检测反应（PCR / LDR），连接依赖性聚合酶 连锁反应（LD-PCR），寡核苷酸连接测定（OLA），扩增连接（LDA），挂锁探针，开环探针和其他可环状探针的连接以及迭代间隙连接（IGL）。