Sequence-specific ligation of DNA using RecA protein.
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.
Ferrin
L J
LJ
Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive, and Kidney Disorders, Building 10, Room 9D20, 10 Center Drive, MSC 1810, Bethesda, MD 20892-1810, USA. lancef@bdg10.niddk.nih.gov
Camerini-Otero
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RD
D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D051518:Fibroblast Growth Factor 3; D005346:Fibroblast Growth Factors / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011938:Rec A Recombinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D004271:DNA, Fungal; D004279:DNA, Viral; C496444:FGF3 protein, human; D051518:Fibroblast Growth Factor 3; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D005346:Fibroblast Growth Factors; D004247:DNA; D011938:Rec A Recombinases