Abstract
End-labeling is a rapid and sensitive method for radioactively, or nonisotopically, labeling DNA fragments and is useful in visualizing small amounts of DNA. End-labeling can also be used to label fragments at one end. All the enzymes employed are specific to either the 3′ or 5′ termini of DNA and will consequently only incorporate a label once per DNA strand. If double-stranded DNA is used, both ends are labeled, but single-end labeled fragments can be produced by further restriction enzyme digestion. This works well with DNA fragments cloned into polylinkers as one labeled end can be removed as a tiny DNA fragment, making subsequent purification easier. Such single-end-labeled molecules can be used to order restriction enzyme fragments and are a prerequisite for Maxam-Gilbert DNA sequencing (1). End-labeled synthetic oligonucleotides have numerous applications, including sequence-specific probes (2); gel retardation, and Southwestern assays (3) and sequencing polymerase chain reaction (PCR) products (4).
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References
Pickersky, E. (1996) Terminal labelling for Maxam-Gilbert sequencing, in Basic DNA and RNA Protocols, Methods in Molecular Biology, vol. 58 (Harwood, A. J., ed.), Humana, Totowa, NJ, pp. 441–446.
Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Hybridisation of synthetic oligodeoxyribonucleotides to φχ174 DNA: the effect of single base pair mismatch. Nucleic Acid Res. 6, 3543–3557.
Scott, V., Clarke, A. R., and Docherty, K. (1994) Protocols for Gene Analysis, Methods in Molecular Biology, vol. 31 (Harwood, A. J., ed.), Humana, Totowa, NJ, pp. 339–345.
Harwood, A. J. and Phear, G. A. (1996) Direct sequencing of PCR products. Basic DNA and RNA Protocols, Methods in Molecular Biology, vol. 58 (Harwood, A. J., ed.), Humana, Totowa, NJ, pp. 403–412.
Klenow, H., Overgaard-Hansen, K., and Patkar, S. A. (1971) Proteolytic cleavage of native DNA polymerase into two different catalytic fragments. Eur. J. Biochem. 22, 371–381.
Challberg, M. D. and Englund, P. T. (1980) Specific labelling of 3′ termini with T4 DNA polymerase. Methods Enzymol. 65, 39–43.
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© 2000 Humana Press Inc., Totowa, NJ
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Harwood, A.J. (2000). End-Labeling of DNA Fragments. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:117
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DOI: https://doi.org/10.1385/1-59259-038-1:117
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
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