公开(公告)号：US08541218B2

公开(公告)日：2013-09-24

申请号：US11167048

申请日：2005-06-24

申请人： Francis Barany ,  Weiguo Cao ,  Jie Tong

发明人： Francis Barany ,  Weiguo Cao ,  Jie Tong

IPC分类号： C12N9/00 ,  C12N15/52

CPC分类号： C12N9/93

摘要： The present invention is directed to a mutant thermostable ligase having substantially higher fidelity than either T4 ligase or Thermus thermophilus ligase. The ligase of the present invention is a mutant of a wild-type thermostable ligase having a histidine adjacent a KXDG motif, where the mutant thermostable ligase has a mutation in its amino sequence where the histidine adjacent the KXDG motif in the wild-type thermostable ligase is replaced with an arginine, and wherein X is any amino acid. The DNA molecule encoding this enzyme as well as expression systems and host cells containing it are also disclosed. The thermostable ligase of the present invention is useful in carrying out a ligase detection reaction process and a ligase chain reaction process.

公开(公告)号：US07960159B2

公开(公告)日：2011-06-14

申请号：US12874255

申请日：2010-09-02

申请人： Francis Barany ,  Weiguo Cao ,  Jianmin Huang ,  Jing Lu

发明人： Francis Barany ,  Weiguo Cao ,  Jianmin Huang ,  Jing Lu

IPC分类号： C12N9/22

CPC分类号： C12Q1/683 ,  C12Q2531/113 ,  C12Q2521/501 ,  C12Q2521/301

摘要： The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

摘要翻译： 本发明是检测DNA序列差异的方法，包括单核苷酸突变或多态性，一个或多个核苷酸插入和一个或多个核苷酸缺失。 制备含有碱基错配的标记的异源双链PCR片段。 核酸内切酶在含有变异（一个或多个错配碱基）的位置和较少程度上在非变体（完全匹配）位置处切割异源双链PCR片段。 用DNA连接酶连接切割产物校正非变体切割，从而大大减少背景。 然后进行检测步骤，其中检测反应产物，并确定序列变异的位置。

公开(公告)号：US07198894B2

公开(公告)日：2007-04-03

申请号：US09998481

申请日：2001-11-30

申请人： Francis Barany ,  Weiguo Cao ,  Jianmin Huang ,  Jing Lu

发明人： Francis Barany ,  Weiguo Cao ,  Jianmin Huang ,  Jing Lu

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/683 ,  C12Q2531/113 ,  C12Q2521/501 ,  C12Q2521/301

摘要： The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

摘要翻译： 本发明是检测DNA序列差异的方法，包括单核苷酸突变或多态性，一个或多个核苷酸插入和一个或多个核苷酸缺失。 制备含有碱基错配的标记的异源双链PCR片段。 核酸内切酶在含有变异（一个或多个错配碱基）的位置和较少程度上在非变体（完全匹配）位置处切割异源双链PCR片段。 用DNA连接酶连接切割产物校正非变体切割，从而大大减少背景。 然后进行检测步骤，其中检测反应产物，并确定序列变异的位置。

公开(公告)号：US07807431B2

公开(公告)日：2010-10-05

申请号：US11682172

申请日：2007-03-05

申请人： Francis Barany ,  Weiguo Cao ,  Jianmin Huang ,  Jing Lu

发明人： Francis Barany ,  Weiguo Cao ,  Jianmin Huang ,  Jing Lu

IPC分类号： C12N9/22

CPC分类号： C12Q1/683 ,  C12Q2531/113 ,  C12Q2521/501 ,  C12Q2521/301

摘要： The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

摘要翻译： 本发明是检测DNA序列差异的方法，包括单核苷酸突变或多态性，一个或多个核苷酸插入和一个或多个核苷酸缺失。 制备含有碱基错配的标记的异源双链PCR片段。 核酸内切酶在含有变异（一个或多个错配碱基）的位置和较少程度上在非变体（完全匹配）位置处切割异源双链PCR片段。 用DNA连接酶连接切割产物校正非变体切割，从而大大减少背景。 然后进行检测步骤，其中检测反应产物，并确定序列变异的位置。

公开(公告)号：US20100323425A1

公开(公告)日：2010-12-23

申请号：US12874255

申请日：2010-09-02

申请人： Francis BARANY ,  Weiguo CAO ,  Jianmin HUANG ,  Jing LU

发明人： Francis BARANY ,  Weiguo CAO ,  Jianmin HUANG ,  Jing LU

IPC分类号： C12N9/16

CPC分类号： C12Q1/683 ,  C12Q2531/113 ,  C12Q2521/501 ,  C12Q2521/301

摘要： The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

摘要翻译： 本发明是检测DNA序列差异的方法，包括单核苷酸突变或多态性，一个或多个核苷酸插入和一个或多个核苷酸缺失。 制备含有碱基错配的标记的异源双链PCR片段。 核酸内切酶在含有变异（一个或多个错配碱基）的位置和较少程度上在非变体（完全匹配）位置处切割异源双链PCR片段。 用DNA连接酶连接切割产物校正非变体切割，从而大大减少背景。 然后进行检测步骤，其中检测反应产物，并确定序列变异的位置。

公开(公告)号：US20080003601A1

公开(公告)日：2008-01-03

申请号：US11682172

申请日：2007-03-05

申请人： Francis BARANY ,  Weiguo CAO ,  Jianmin HUANG ,  Jing LU

发明人： Francis BARANY ,  Weiguo CAO ,  Jianmin HUANG ,  Jing LU

IPC分类号： C12Q1/68 ,  C12N9/14

CPC分类号： C12Q1/683 ,  C12Q2531/113 ,  C12Q2521/501 ,  C12Q2521/301

摘要： The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.

摘要翻译： 本发明是检测DNA序列差异的方法，包括单核苷酸突变或多态性，一个或多个核苷酸插入和一个或多个核苷酸缺失。 制备含有碱基错配的标记的异源双链PCR片段。 核酸内切酶在含有变异（一个或多个错配碱基）的位置和较少程度上在非变体（完全匹配）位置处切割异源双链PCR片段。 用DNA连接酶连接切割产物校正非变体切割，从而大大减少背景。 然后进行检测步骤，其中检测反应产物，并确定序列变异的位置。

公开(公告)号：US06949370B1

公开(公告)日：2005-09-27

申请号：US09830502

申请日：1999-10-29

申请人： Francis Barany ,  Weiguo Cao ,  Jie Tong

发明人： Francis Barany ,  Weiguo Cao ,  Jie Tong

IPC分类号： C07H21/04 ,  C12N9/00 ,  C12N9/22 ,  C12Q1/68

CPC分类号： C12N9/93

摘要： The present invention is directed to a thermostable ligase having substantially higher fidelity than either T4 ligase or Thermus thermophilus ligase. The DNA molecule encoding this enzyme as well as expression systems and host cells containing it are also disclosed. The thermostable ligase of the present invention is useful in carrying out a ligase detection reaction process and a ligase chain reaction process.

摘要翻译： 本发明涉及一种具有比T4连接酶或Thermus thermophilus连接酶具有更高保真度的热稳定连接酶。 还公开了编码该酶的DNA分子以及含有它的表达系统和宿主细胞。 本发明的热稳定连接酶可用于进行连接酶检测反应过程和连接酶链反应过程。

公开(公告)号：US20050266487A1

公开(公告)日：2005-12-01

申请号：US11167048

申请日：2005-06-24

申请人： Francis Barany ,  Weiguo Cao ,  Jie Tong

发明人： Francis Barany ,  Weiguo Cao ,  Jie Tong

IPC分类号： C07H21/04 ,  C12N9/00 ,  C12N9/22 ,  C12Q1/68

CPC分类号： C12N9/93

摘要： The present invention is directed to a mutant thermostable ligase having substantially higher fidelity than either T4 ligase or Thermus thermophilus ligase. The ligase of the present invention is a mutant of a wild-type thermostable ligase having a histidine adjacent a KXDG motif, where the mutant thermostable ligase has a mutation in its amino sequence where the histidine adjacent the KXDG motif in the wild-type thermostable ligase is replaced with an arginine, and wherein X is any amino acid. The DNA molecule encoding this enzyme as well as expression systems and host cells containing it are also disclosed. The thermostable ligase of the present invention is useful in carrying out a ligase detection reaction process and a ligase chain reaction process.

摘要翻译： 本发明涉及一种具有比T4连接酶或Thermus thermophilus连接酶具有更高保真度的突变热稳定连接酶。 本发明的连接酶是具有与KXDG基序相邻的组氨酸的野生型热稳定连接酶的突变体，其中突变热稳定连接酶在其氨基序列中具有突变，其中野生型热稳定连接酶中KXDG基序相邻的组氨酸 被精氨酸代替，其中X是任何氨基酸。 还公开了编码该酶的DNA分子以及含有它的表达系统和宿主细胞。 本发明的热稳定连接酶可用于进行连接酶检测反应过程和连接酶链反应过程。