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PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

ABSTRACT: 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 Taq 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.

SUBMITTER: Barnes WM 

PROVIDER: S-EPMC43341 | biostudies-other | 1994 Mar

REPOSITORIES: biostudies-other

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PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

Barnes W M WM  

Proceedings of the National Academy of Sciences of the United States of America 19940301 6

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 Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent).  ...[more]