Sequence-specific ligation of DNA using RecA protein
Autor: | R D Camerini-Otero, L J Ferrin |
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Rok vydání: | 1998 |
Předmět: |
DNA
Complementary Base pair DNA polymerase II Fibroblast Growth Factor 3 Genetic Vectors Saccharomyces cerevisiae Polymerase cycling assembly Sticky and blunt ends Proto-Oncogene Proteins Escherichia coli Humans Cloning Molecular DNA Fungal chemistry.chemical_classification DNA ligase Binding Sites Multidisciplinary DNA clamp Base Sequence biology DNA Biological Sciences Bacteriophage lambda Molecular biology Fibroblast Growth Factors Rec A Recombinases Oligodeoxyribonucleotides chemistry DNA Viral biology.protein DNA polymerase I In vitro recombination |
Zdroj: | Proceedings of the National Academy of Sciences. 95:2152-2157 |
ISSN: | 1091-6490 0027-8424 |
Popis: | A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR. |
Databáze: | OpenAIRE |
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