Topic
DNA ligase
About: DNA ligase is a research topic. Over the lifetime, 6418 publications have been published within this topic receiving 347354 citations.
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TL;DR: An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.
Abstract: We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
8,117 citations
TL;DR: A simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine, based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent.
Abstract: We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.
5,445 citations
TL;DR: Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase.
3,339 citations
TL;DR: It is proposed that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I, which results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNATopoisomersase I.
2,254 citations
TL;DR: It is shown that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4, illustrating that HR and NHEJ compete to process DNA breaks that arise during DNA replication.
1,482 citations