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。