A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus.-猫眼课题宝

使用来自黄栖热菌的热稳定 DNA 聚合酶的快速聚合酶链反应定向测序策略。

DOI：

10.1016/0378-1119(92)90665-c

发表时间：

1992

期刊：

Gene

影响因子：

3.5

通讯作者：

中科院分区：

生物学3区

文献类型：

作者：

V. Rao;N. B. Saunders

我们开发了一种聚合酶链反应（PCR）定向测序策略，用于从粗病毒或细胞制剂中快速测序DNA。基本策略包括两个阶段。在第一阶段，用低浓度的脱氧核糖核苷酸三磷酸（dNTP）和寡脱氧核糖核苷酸引物进行对称PCR扩增目标DNA。结果是DNA在初始循环中呈指数级扩增，由于dNTP和引物的浓度有限，在25个循环中达到平台期。在第二阶段，在5 '标记引物和四种二脱氧核糖核苷酸三磷酸之一的存在下，通过不对称PCR扩增一小部分PCR混合物，而不进行任何纯化。这导致单链DNA产物的积累，通过结合适当的二脱氧核糖核苷酸单磷酸在特定点终止。然后通过测序凝胶电泳分析产物，然后进行放射自显影。PCR条件经过优化，可以在一到两天内产生数百个核苷酸的序列阶梯，从低至100个噬菌体或细菌基因组拷贝开始。

We have developed a polymerase chain reaction (PCR)-directed sequencing strategy for rapid sequencing of DNA from crude viral or cell preparations. The basic strategy consists of two phases. In the first phase, the target DNA is amplified by symmetric PCR with low concentrations of deoxyribonucleotide triphosphate (dNTP) and oligodeoxyribonucleotide primers. This results is exponential amplification of DNA in the initial cycles, reaching a plateau by 25 cycles due to limiting concentrations of dNTP and primers. In the second phase, a small aliquot of the PCR mixture is amplified without any purification, by asymmetric PCR in the presence of a 5′-labeled primer and one of the four dideoxyribonucleotide triphosphates. This results in the accumulation of single-stranded DNA products that are terminated at specific points by incorporation of the appropriate dideoxyribonucleotide monophosphate. The products are then analyzed by electrophoresis on a sequencing gel followed by autoradiography. The PCR conditions are optimized to generate sequence ladders of several hundred nucleotides starting from as low as 100 copies of bacteriophage or bacterial genome in one to two days.

Coupled amplification and sequencing of genomic DNA.