Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome.
Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome.
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DOI：
10.1093/nar/gkl547
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发表时间：
2006-08-07
影响因子：
14.9
通讯作者：
Kong H
中科院分区：
文献类型：
--
作者：
Xu Y;Kim HJ;Kays A;Rice J;Kong H
本研究描述了一种新的解旋酶介导的等温DNA扩增方法，以指数方式扩增环状DNA。环状解旋酶依赖性扩增（cHDA）系统基于T7复制机制，包括进行性T7解旋酶、核酸外切酶缺陷型T7 DNA聚合酶（T7测序酶）和T7 Gp2.5单链DNA结合（SSB）蛋白。双链体DNA模板被T7解旋酶解旋后，特异性引物与分离的DNA链退火，T7测序酶通过滚环机制延伸每个引物的3′端，不仅扩增引物限定的区域，还扩增模板的连续多联体。cHDA反应可以在一个温度（25°C）下进行整个过程，并且可以实现高达10000倍的扩增。扩增可以使用纯化的质粒DNA或粗细胞裂解物进行，并且可以扩增大至10 kb的插入物。cHDA反应后，扩增产物可直接用于测序和限制性酶消化，无需进一步纯化。利用该解旋酶，可以在一个恒温条件下，一步简单地同时筛选和扩增环状DNA样品。
This study describes a novel helicase-mediated isothermal DNA amplification method that exponentially amplifies circular DNAs. The circular helicase-dependent amplification (cHDA) system is based on the T7 replication machinery, which includes the processive T7 helicase, an exonuclease-deficient T7 DNA polymerase (T7 Sequenase) and the T7 Gp2.5 single-stranded DNA-binding (SSB) protein. After the duplex DNA template is unwound by the T7 helicase, specific primers anneal to the separated DNA strands and T7 Sequenase extends the 3′ end of each primer by a rolling circle mechanism to amplify not only a region defined by the primers but also continuous concatemers of the template. The cHDA reaction can be carried out at one temperature (25°C) for the entire process and can achieve up to 10 000-fold amplification. Amplification can be performed using purified plasmid DNA or a crude cell lysate and can amplify inserts as large as 10 kb. Following a cHDA reaction, the amplified products can be used directly for sequencing and restriction enzyme digestion without further purification. By utilizing the helicase enzyme, circular DNA samples can be simultaneously screened and amplified at one constant temperature in one easy step.
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影响因子：
14.9
作者：
MATTILA, P;KORPELA, J;PITKANEN, K
通讯作者：
PITKANEN, K
DOI：
10.1007/bf00271243
发表时间：
1973-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
SCHERZINGER, E;LITFIN, F;JOST, E
通讯作者：
JOST, E
影响因子：
4.8
作者：
Notarnicola, SM;Mulcahy, HL;Richardson, CC
通讯作者：
Richardson, CC
DOI：
10.1073/pnas.171317698
发表时间：
2001-08-14
影响因子：
11.1
作者：
Hollis, T;Stattel, JM;Ellenberger, T
通讯作者：
Ellenberger, T
影响因子：
5.6
作者：
DUNN, JJ;STUDIER, FW
通讯作者：
STUDIER, FW