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Poly nucleotide kinase exchange reaction quantitative assay for restriction endo nuclease generated 5 phosphoryl termini in dna

Poly nucleotide kinase exchange reaction quantitative assay for restriction endo nuclease generated 5 phosphoryl termini in dna

Journal of Biological Chemistry 252(10): 3176-3184

A rapid, quantitative assay for measuring the cleavage of duplex DNA by restriction endonucleases was developed. The assay employs phage T4 polynucleotide kinase-mediated exchange of [.gamma.-32]ATP with the 5'-phosphoryl termini generated by type II restriction enzymes. Parameters of the reaction were studied, using EcoRI endonuclease digested .lambda. DNA as a substrate. The optimal ATP concentration (10 .mu.M) and pH (6.6) are similar to those values reported for polynucleotide kinase-mediated exchange using single-stranded oligonucleotides as substrates. The ADP concentration required for optimal exchange (300 .mu.M) is considerably higher. Using conditions optimized for the exchange of [.gamma.-32]ATP with the 5'-phosphoryl group of EcoRI termini, the utility of the exchange reaction as an assay for restriction endonuclease cleavage of DNA was studied. Incorporation of 32Pi into DNA termini is dependent on the amount of polynucleotide kinase employed, and with moderate levels of enzyme exchange is incomplete. This may reflect the presence of significant quantities of enzyme .cntdot. DNA complexes having unexchanged 5'-phosphoryl termini. Nevertheless, this does not invalidate the assay. For a given amount of polynucleotide kinase, the extent of exchange is reproducible and proportional to the number of DNA termini that are present. Fragments of DNA varying from 4 .times. 105-14 .times. 106 daltons are equally well labeled, and the termini generated by EcoRI cleavage are labeled 30 times more efficiently than the termini at internal nicks. The assay was used to follow the EcoRI endonuclease cleavage of polyoma [virus] DNA. The rate of formation of Form III DNA is the same when measured by quantitation of the amount of Form III DNA present at different times, or by measuring the extent of incorporation of 32Pi into the duplex termini.

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