PCR AMPLIFICATION OF UP TO 35-KB DNA WITH HIGH-FIDELITY AND HIGH-YIELD FROM LAMBDA-BACTERIOPHAGE TEMPLATES
PCR AMPLIFICATION OF UP TO 35-KB DNA WITH HIGH-FIDELITY AND HIGH-YIELD FROM LAMBDA-BACTERIOPHAGE TEMPLATES
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DOI：
10.1073/pnas.91.6.2216
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发表时间：
1994-03-15
影响因子：
11.1
通讯作者：
BARNES, WM
中科院分区：
文献类型：
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作者：
BARNES, WM
DNA的PCR扩增的靶长度限制已经被鉴定和解决。与此同时，碱基对的保真度，使用PCR产物作为引物的能力，和目标片段的最大产量增加。这些改进是通过组合高水平的无核酸外切酶的Tag DNA聚合酶的N-末端缺失突变体Klentaq 1与非常低水平的表现出3 '-核酸外切酶活性的热稳定DNA聚合酶（Pfu、Vent或Deep Vent）来实现的。至少35 kb可以从1 ng的λ DNA模板扩增到高产率。
A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Tag DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.