Method for sequencing nucleic acid molecules

    公开(公告)号:US07056676B2

    公开(公告)日:2006-06-06

    申请号:US11015138

    申请日:2004-12-16

    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.

    Method for sequencing nucleic acid molecules
    55.
    发明申请
    Method for sequencing nucleic acid molecules 有权
    核酸分子测序方法

    公开(公告)号:US20050202466A1

    公开(公告)日:2005-09-15

    申请号:US11015138

    申请日:2004-12-16

    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。

    Method for sequencing nucleic acid molecules

    公开(公告)号:US20050164255A1

    公开(公告)日:2005-07-28

    申请号:US11013578

    申请日:2004-12-15

    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.

    NANOPORES IN ZERO MODE WAVEGUIDES
    59.
    发明申请
    NANOPORES IN ZERO MODE WAVEGUIDES 有权
    纳秒在零模式波形

    公开(公告)号:US20130240356A1

    公开(公告)日:2013-09-19

    申请号:US13669186

    申请日:2012-11-05

    IPC分类号: G01N27/447 G02B6/00

    摘要: Methods, devices, substrates, and systems are disclosed involving arrays of zero-mode waveguides having nanopores extending through the bases that form the bottoms of the zero-mode-waveguides. Electric fields across the nanopores are used to attach single biomolecules such as polymerase enzymes within each zero-mode-waveguide. Electric fields across the nanopores can also be used for the active loading of nucleic acid templates into enzymes attached within the zero mode waveguides.

    摘要翻译: 公开的方法,装置,衬底和系统涉及具有延伸穿过形成零模式波导的底部的基底的纳米孔的零模式波导阵列。 纳米孔中的电场用于在每个零模式波导内附着单个生物分子,如聚合酶。 纳米孔中的电场也可用于将核酸模板活性负载加载到零模式波导内附着的酶中。