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公开(公告)号：US20110111401A1
公开(公告)日：2011-05-12
申请号：US12841819
申请日：2010-07-22
申请人： Jonas KORLACH , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas KORLACH , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
摘要翻译： 本发明涉及对具有多个碱基的靶核酸分子进行测序的方法。 在其原理中，聚合反应中碱添加的时间顺序是在核酸分子上测量的，即核酸聚合酶在待测序的模板核酸分子上的活性被实时跟踪。 通过在碱添加序列的每个步骤中通过核酸聚合酶的催化活性鉴定哪个碱基被掺入靶核酸的生长互补链中来推断该序列。 在靶核酸分子复合物上提供聚合酶，其适于沿着靶核酸分子移动并在活性位点延伸寡核苷酸引物。 多个标记类型的核苷酸类似物在活性位点附近提供，每种可区分类型的核苷酸类似物与靶核酸序列中的不同核苷酸互补。 生长的核酸链通过使用聚合酶延伸到活性位点处的核酸链的核苷酸类似物，其中加入的核苷酸类似物与活性位点上的靶核酸的核苷酸互补。 鉴定作为聚合步骤的结果添加到寡核苷酸引物中的核苷酸类似物。 重复提供标记的核苷酸类似物，聚合生长的核酸链和鉴定添加的核苷酸类似物的步骤，使得核酸链进一步延长并确定靶核酸的序列。
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公开(公告)号：US07056676B2
公开(公告)日：2006-06-06
申请号：US11015138
申请日：2004-12-16
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
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公开(公告)号：US07943305B2
公开(公告)日：2011-05-17
申请号：US11279711
申请日：2006-04-13
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid. Each type of labeled nucleotide comprises an acceptor fluorophore attached to a phosphate portion of the nucleotide such that the fluorophore is removed upon incorporation into a growing strand. Fluorescent signal is emitted via fluorescent resonance energy transfer between the donor fluorophore and the acceptor fluorophore as each nucleotide is incorporated into the growing strand. The sequence is deduced by identifying which base is being incorporated into the growing strand.
摘要翻译： 本发明涉及对具有多个碱基的靶核酸分子进行测序的方法。 在其原理中，聚合反应中碱添加的时间顺序是在核酸分子上测量的。 每种类型的标记核苷酸包括连接到核苷酸的磷酸部分的受体荧光团，使得在掺入生长链中时除去荧光团。 当每个核苷酸并入生长链中时，通过供体荧光团和受体荧光团之间的荧光共振能量转移发射荧光信号。 通过鉴定哪个碱基被结合到生长链中推导出序列。
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公开(公告)号：US20090137007A1
公开(公告)日：2009-05-28
申请号：US12265616
申请日：2008-11-05
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
摘要翻译： 本发明涉及对具有多个碱基的靶核酸分子进行测序的方法。 在其原理中，聚合反应中碱添加的时间顺序是在核酸分子上测量的，即核酸聚合酶在待测序的模板核酸分子上的活性被实时跟踪。 通过在碱添加序列的每个步骤中通过核酸聚合酶的催化活性鉴定哪个碱基被掺入靶核酸的生长互补链中来推断该序列。 在靶核酸分子复合物上提供聚合酶，其适于沿着靶核酸分子移动并在活性位点延伸寡核苷酸引物。 多个标记类型的核苷酸类似物在活性位点附近提供，每种可区分类型的核苷酸类似物与靶核酸序列中的不同核苷酸互补。 生长的核酸链通过使用聚合酶延伸到活性位点处的核酸链的核苷酸类似物，其中加入的核苷酸类似物与活性位点上的靶核酸的核苷酸互补。 鉴定作为聚合步骤的结果添加到寡核苷酸引物中的核苷酸类似物。 重复提供标记的核苷酸类似物，聚合生长的核酸链和鉴定添加的核苷酸类似物的步骤，使得核酸链进一步延长并确定靶核酸的序列。
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公开(公告)号：US20080227654A1
公开(公告)日：2008-09-18
申请号：US12027242
申请日：2008-02-06
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonuelcotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymeras to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
摘要翻译： 本发明涉及对具有多个碱基的靶核酸分子进行测序的方法。 在其原理中，聚合反应中碱添加的时间顺序是在核酸分子上测量的，即核酸聚合酶在待测序的模板核酸分子上的活性被实时跟踪。 通过在碱添加序列的每个步骤中通过核酸聚合酶的催化活性鉴定哪个碱基被掺入靶核酸的生长互补链中来推断该序列。 提供了靶核酸分子复合物上的聚合酶，其适于沿着靶核酸分子移动并在活性位点延伸寡核苷酸引物。 多个标记类型的核苷酸类似物在活性位点附近提供，每种可区分类型的核苷酸类似物与靶核酸序列中的不同核苷酸互补。 生长的核酸链通过使用聚合物将核苷酸类似物添加到活性位点处的核酸链延伸，其中加入的核苷酸类似物与活性位点处的靶核酸的核苷酸互补。 鉴定作为聚合步骤的结果添加到寡核苷酸引物中的核苷酸类似物。 重复提供标记的核苷酸类似物，聚合生长的核酸链和鉴定添加的核苷酸类似物的步骤，使得核酸链进一步延长并确定靶核酸的序列。
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公开(公告)号：US07056661B2
公开(公告)日：2006-06-06
申请号：US09572530
申请日：2000-05-17
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
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公开(公告)号：US07033764B2
公开(公告)日：2006-04-25
申请号：US11014015
申请日：2004-12-15
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
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公开(公告)号：US07485424B2
公开(公告)日：2009-02-03
申请号：US11277114
申请日：2006-03-21
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
摘要翻译： 本发明涉及对具有多个碱基的靶核酸分子进行测序的方法。 在其原理中，聚合反应中碱添加的时间顺序是在核酸分子上测量的，即核酸聚合酶在待测序的模板核酸分子上的活性被实时跟踪。 通过在碱添加序列的每个步骤中通过核酸聚合酶的催化活性鉴定哪个碱基被掺入靶核酸的生长互补链中来推断该序列。 在靶核酸分子复合物上提供聚合酶，其适于沿着靶核酸分子移动并在活性位点延伸寡核苷酸引物。 多个标记类型的核苷酸类似物在活性位点附近提供，每种可区分类型的核苷酸类似物与靶核酸序列中的不同核苷酸互补。 生长的核酸链通过使用聚合酶延伸到活性位点处的核酸链的核苷酸类似物，其中加入的核苷酸类似物与活性位点上的靶核酸的核苷酸互补。 鉴定作为聚合步骤的结果添加到寡核苷酸引物中的核苷酸类似物。 重复提供标记的核苷酸类似物，聚合生长的核酸链和鉴定添加的核苷酸类似物的步骤，使得核酸链进一步延长并确定靶核酸的序列。
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公开(公告)号：US07052847B2
公开(公告)日：2006-05-30
申请号：US11013578
申请日：2004-12-15
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
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公开(公告)号：US07943307B2
公开(公告)日：2011-05-17
申请号：US11336122
申请日：2006-01-19
申请人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
发明人： Jonas Korlach , Watt W. Webb , Michael Levene , Stephen Turner , Harold G. Craighead , Mathieu Foquet
CPC分类号： C12Q1/6874 , C12Q1/6869 , Y10S436/80 , Y10S436/805 , Y10T436/143333 , C12Q2565/537 , C12Q2537/149 , C12Q2561/113 , C12Q2521/543 , C12Q2565/518 , C12Q2561/12
摘要： The present invention is directed to a method of sequencing a target nucleic acid. The method provides a complex comprising a polymerase enzyme, a target nucleic acid molecule, and a primer, wherein the complex is immobilized on a support Fluorescent label is attached to a terminal phosphate group of the nucleotide or nucleotide analog. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The time duration of the signal from labeled nucleotides or nucleotide analogs that become incorporated is distinguished from freely diffusing labels by a longer retention in the observation volume for the nucleotides or nucleotide analogs that become incorporated than for the freely diffusing labels.
摘要翻译： 本发明涉及对靶核酸进行测序的方法。 该方法提供了一种包含聚合酶，靶核酸分子和引物的复合物，其中复合物被固定在载体上荧光标记附着于核苷酸或核苷酸类似物的末端磷酸基团。 通过使用聚合酶将核酸类似物加入到核酸链中来延长生长的核酸链。 鉴定作为聚合步骤的结果添加到寡核苷酸引物中的核苷酸类似物。 来自被引入的标记核苷酸或核苷酸类似物的信号的持续时间与自由扩散标记的区别在于，对于被掺入的核苷酸或核苷酸类似物的观察体积比对于自由扩散标记物的观察体积更长的保留。
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