Efficient long-PCR site-specific mutagenesis of a high GC template
| Autor: | Galina Rybachuk, Konstantin G. Kousoulas, Sukhanya Jayachandra, Vladimir N. Chouljenko |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Guanine
Genes Viral DNA Recombinant Mutagenesis (molecular biology technique) DNA-Directed DNA Polymerase Biology Recombinant virus medicine.disease_cause Polymerase Chain Reaction General Biochemistry Genetics and Molecular Biology law.invention chemistry.chemical_compound Cytosine Viral Proteins law medicine Simplexvirus Site-directed mutagenesis Deoxyribonucleases Type II Site-Specific Gene Polymerase chain reaction Viral Structural Proteins Mutation Templates Genetic Molecular biology chemistry Mutagenesis Site-Directed DNA GC-content Biotechnology |
| Zdroj: | BioTechniques. 21(3) |
| ISSN: | 0736-6205 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|