公开(公告)号：US5981186A

公开(公告)日：1999-11-09

申请号：US892003

申请日：1997-07-14

申请人： Chris Gabe ,  Rodney D. Gilchrist

发明人： Chris Gabe ,  Rodney D. Gilchrist

IPC分类号： C12Q1/68 ,  G01N27/447 ,  C12P19/34

CPC分类号： G01N27/44726 ,  C12Q1/6869

摘要： The sequence of a target nucleic acid polymer can be determined by (a) performing a first chain-extension sequencing reaction on the target nucleic acid polymer in a reaction mixture containing first and second chain-terminators to produce a first product mixture containing commonly-labeled polynucleotide fragments complementary to a first strand of the target nucleic acid polymer, each fragment in the mixture being terminated with the first or second chain-terminator; (b) performing a second chain extension sequencing reaction on the target nucleic acid polymer in a reaction mixture containing the first and a third chain-terminator to produce a second product mixture containing commonly-labeled polynucleotide fragments complementary to the first strand of the target nucleic acid polymer, each fragment in the mixture being terminated with the first or the third chain-terminator, said first, second and third chain-terminators each being different; and (c) evaluating the lengths of the polynucleotide fragments in the first and second product mixtures to determine the sequence of the target nucleic acid polymer. In the evaluation step, the first and second product mixtures can be evaluated in two separate lanes of a gel, in which case the labels employed in the two chain extension sequencing reactions can be the same. Alternatively, if the labels employed in the two chain extension reactions are different and spectroscopically distinguishable from one another, the first and second product mixtures can be combined before electrophoresis and the entire analysis can be performed in a single lane of a gel.

摘要翻译： 靶核酸聚合物的序列可以通过（a）在含有第一和第二链终止剂的反应混合物中对目标核酸聚合物进行第一链延伸测序反应来测定，以产生含有常规标记的第一产物混合物 与靶核酸聚合物的第一链互补的多核苷酸片段，混合物中的每个片段用第一或第二链终止子终止; （b）在含有第一和第三链终止子的反应混合物中对目标核酸聚合物进行第二链延伸测序反应以产生含有与靶核酸的第一链互补的通常标记的多核苷酸片段的第二产物混合物 所述混合物中的每个片段用第一或第三链终止剂终止，所述第一，第二和第三链终止剂各自不同; 和（c）评估第一和第二产物混合物中的多核苷酸片段的长度以确定靶核酸聚合物的序列。 在评估步骤中，第一和第二产物混合物可以在两个独立的凝胶通道中进行评估，在这种情况下，两个延伸测序反应中使用的标记可以相同。 或者，如果两个延伸反应中使用的标记是不同的并且在光谱上彼此区分，则可以在电泳之前组合第一和第二产物混合物，并且可以在凝胶的单一泳道中进行整个分析。