Assay of RNA-linked nascent DNA pieces with polynucleotide kinase. |
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Authors: | R Okazaki S Hirose T Okazaki T Ogawa Y Kurosawa |
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Affiliation: | Institute of Molecular Biology Faculty of Science, Nagoya University Nagoya, Japan 464 |
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Abstract: | The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-32P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type. |
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Keywords: | 5′-OH DNA 5′-hydroxyl terminated DNA 5′-P DNA 5′-phosphoryl terminated DNA |
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