Solid phase in vitro mutagenesis using plasmid DNA template.
AUTOR(ES)
Hultman, T
RESUMO
Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=332130Documentos Relacionados
- In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.
- Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template
- Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.
- Template functions in the enzymic formation of polyribonucleotides. IV. Denatured DNA as template.
- Heat shock-regulated transcription in vitro from a reconstituted chromatin template.