A RCA-based assay for analyzing individual strand break in DNA heteroduplex cleavage by restriction endonucleases.
نویسندگان
چکیده
We have developed a rapid and high-throughput assay based on rolling circle amplification, to distinguish individual strand cleavage of DNA duplexes by restriction endonucleases. As an illustration, we analyzed nicking activity of Nb.BbvCI and uneven cleavage of LNA modified DNA by EcoRI. This assay has potential for analyzing protein-DNA interactions.
منابع مشابه
Cleavage of mispaired heteroduplex DNA substrates by numerous restriction enzymes.
The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction en...
متن کاملCleavage of Mispaired Heteroduplex DNA Substrates by Numerous Restriction Enzymes 1 Cleavage of mispaired heteroduplex DNA substrates by numerous restriction enzymes
The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction en...
متن کاملSequence-specific cleavage of RNA by Type II restriction enzymes
The ability of 223 Type II restriction endonucleases to hydrolyze RNA-DNA heteroduplex oligonucleotide substrates was assessed. Despite the significant topological and sequence asymmetry introduced when one strand of a DNA duplex is substituted by RNA we find that six restriction enzymes (AvaII, AvrII, BanI, HaeIII, HinfI and TaqI), exclusively of the Type IIP class that recognize palindromic o...
متن کاملTarget site cleavage by the monomeric restriction enzyme BcnI requires translocation to a random DNA sequence and a switch in enzyme orientation
Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both DNA strands after th...
متن کاملStrand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.
A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dy...
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ورودعنوان ژورنال:
- Chemical communications
دوره 50 80 شماره
صفحات -
تاریخ انتشار 2014