End-Labeling of DNA Fragments
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).
KeywordsRestriction Enzyme Site Calf Intestinal Alkaline Phosphatase Sequencing Polymerase Chain Reaction Deoxyuridine Triphosphate Label Nucleotide
- 3.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.Google Scholar