Chapter title |
Radiolabeling of DNA by Nick Translation
|
---|---|
Book title |
Nucleic Acids
|
Published in |
Methods in molecular biology, December 1984
|
DOI | 10.1385/0-89603-064-4:257 |
Pubmed ID | |
Book ISBNs |
978-0-89603-064-0, 978-1-59259-489-4
|
Authors |
Mathew, C. G. P., C. G. P. Mathew |
Abstract |
Nick translation is the name given to a reaction that is used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2). Free 3'-hydroxyl groups are created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse 1). DNA polymerase 1 from E. coli will then catalyze the addition of a nucleotide residue to the 3'-hydroxyl terminus of the nick. At the same time, the 5'- to 3'-exonuclease activity of this enzyme will eliminate the nucleotide unit from the 5'-phosphoryl terminus of the nick. Thus a new nucleotide with a free 3'-OH group will have been incorporated at the position where the original nucleotide was excised, and the nick will have been shifted along by one nucleotide unit in a 3' direction. This 3' shift, or translation, of the nick will result in the sequential addition of new nucleotides to the DNA while the pre-existing nucleotides will be removed. If radioactively labeled deoxyribonucleoside triphosphates are used as substrates, up to 50% of the residues in the DNA can be labeled.Furthermore, Rigby et al. have shown (2) that the DNA is labeled throughout at a uniform specific activity, which is an important requirement if the DNA is to be used as a probe in molecular hybridization experiments. |
Mendeley readers
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Unknown | 12 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Ph. D. Student | 2 | 17% |
Student > Bachelor | 2 | 17% |
Professor | 1 | 8% |
Student > Master | 1 | 8% |
Unknown | 6 | 50% |
Readers by discipline | Count | As % |
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Unknown | 6 | 50% |