公开(公告)号：US07374885B2

公开(公告)日：2008-05-20

申请号：US11213519

申请日：2005-08-26

申请人： Michael M. Becker ,  Wai-Chung Lam ,  Kristin W. Livezey ,  Steven T. Brentano ,  Daniel P. Kolk ,  Astrid R. W. Schroder

发明人： Michael M. Becker ,  Wai-Chung Lam ,  Kristin W. Livezey ,  Steven T. Brentano ,  Daniel P. Kolk ,  Astrid R. W. Schroder

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12P19/34 ,  C12Q1/6865 ,  C12Q2537/163 ,  C12Q2521/325 ,  C12Q2521/107 ,  C12Q2525/186 ,  C12Q2533/101 ,  C12Q2525/143

摘要： The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the “priming oligonucleotide,” a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products. The method of the present invention minimizes or substantially eliminates the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or substantially eliminates this problem, thus providing an enhanced level of sensitivity.

摘要翻译： 本发明涉及合成目标核酸序列的多个拷贝的新型方法，所述目标核酸序列是自动催化的（即，能够自动循环而不需要修饰诸如温度，pH或离子强度的反应条件，并且使用一种 循环在下一个）。 特别地，本发明公开了一种鲁棒有效的核酸扩增方法，同时减少副产物的外观。 该方法仅使用一种引物，即引物寡核苷酸，经修饰的启动子寡核苷酸，用于防止聚合酶从其3'末端延伸，以及任选的终止引物延伸反应的手段，以在体外扩增RNA或DNA分子，同时 减少或基本上消除副产物的形成。 本发明的方法使副产物的出现最小化或基本上消除，从而提供高水平的特异性。 此外，副产物的出现可能通过各种分子检测技术使扩增反应的分析复杂化。 本发明使这个问题最小化或基本消除，从而提供了增强的灵敏度。

公开(公告)号：US07696337B2

公开(公告)日：2010-04-13

申请号：US11574307

申请日：2005-08-26

申请人： Michael M. Becker ,  Steven T. Brentano ,  Daniel P. Kolk ,  Wai-Chung Lam ,  Kristin W. Livezey ,  Norman C. Nelson ,  Astrid R. W. Schroder ,  Gary P. Schroth

发明人： Michael M. Becker ,  Steven T. Brentano ,  Daniel P. Kolk ,  Wai-Chung Lam ,  Kristin W. Livezey ,  Norman C. Nelson ,  Astrid R. W. Schroder ,  Gary P. Schroth

IPC分类号： C07H21/04 ,  C12Q1/68

CPC分类号： C12P19/34 ,  C12Q1/6865 ,  C12Q2537/163 ,  C12Q2521/325 ,  C12Q2521/107 ,  C12Q2525/186 ,  C12Q2533/101 ,  C12Q2525/143

摘要： The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side products. The method uses only one primer, the “priming oligonucleotide,” a 3′blocked promoter oligonucleotide and optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or eliminating the formation of side products. The method of the present invention minimizes or eliminates the emergence of side products, thus providing a high level of specificity. Furthermore, the appearance of side products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or eliminates this problem, thus providing an enhanced level of sensitivity.

摘要翻译： 本发明涉及合成目标核酸序列的多个拷贝的新型方法，所述目标核酸序列是自动催化的（即，能够自动循环而不需要修饰诸如温度，pH或离子强度的反应条件，并且使用一种 循环在下一个）。 特别地，本发明公开了一种鲁棒有效的核酸扩增方法，同时减少了副产物的外观。 该方法仅使用一种引物，即引物寡核苷酸，3'封闭的启动子寡核苷酸和任选的终止引物延伸反应的手段，以体外扩增RNA或DNA分子，同时减少或消除副产物的形成。 本发明的方法使副产物的出现最小化或消除，从而提供高水平的特异性。 此外，副产物的出现可能通过各种分子检测技术使扩增反应的分析复杂化。 本发明使这个问题最小化或消除了这一点，从而提供了增强的灵敏度。