Efficient long-PCR site-specific mutagenesis of a high GC template

A long PCR method was developed for the efficient site-specific mutagenesis of herpes simplex virus (HSV-1) DNA fragments with high GC content. In this protocol, a PCR product was partially extended first using a cloned DNA fragment. The final mutagenized fragment was produced after a second extensi...

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Veröffentlicht in:BioTechniques. - 1993. - 21(1996), 3 vom: 15. Sept., Seite 472-4, 476-8, 480
1. Verfasser: Chouljenko, V (VerfasserIn)
Weitere Verfasser: Jayachandra, S, Rybachuk, G, Kousoulas, K G
Format: Aufsatz
Sprache:English
Veröffentlicht: 1996
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. DNA, Recombinant UL53 protein, Human herpesvirus 1 Viral Proteins Viral Structural Proteins Guanine 5Z93L87A1R Cytosine mehr... 8J337D1HZY DNA polymerase, Thermus thermophilus EC 2.7.7.- Tli polymerase DNA-Directed DNA Polymerase EC 2.7.7.7 endodeoxyribonuclease SnaBI EC 3.1.21.- Deoxyribonucleases, Type II Site-Specific EC 3.1.21.4
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520 |a A long PCR method was developed for the efficient site-specific mutagenesis of herpes simplex virus (HSV-1) DNA fragments with high GC content. In this protocol, a PCR product was partially extended first using a cloned DNA fragment. The final mutagenized fragment was produced after a second extension using another PCR product and final amplification using external primers. The sequential use of two extension reactions increased the predicted frequency of the engineered mutation in the final product to 100%. This method was used to generate a mutated glycoprotein K (gK) gene specified by HSV-1. A recombinant virus that carried the mutated gK gene caused extensive cell fusion of infected cells 
650 4 |a Journal Article 
650 4 |a Research Support, Non-U.S. Gov't 
650 4 |a Research Support, U.S. Gov't, P.H.S. 
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700 1 |a Kousoulas, K G  |e verfasserin  |4 aut 
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