Box 4.1DNA labeling

Attachment of radioactive, fluorescent or other types of marker to DNA molecules.

DNA labeling is a central part of many molecular biology procedures, including Southern hybridization (Section 4.1.2), fluorescent in situ hybridization (FISH; Section 5.3.2) and DNA sequencing (Section 6.1). It enables the location of a particular DNA molecule - on a nitrocellulose or nylon membrane, in a chromosome or in a gel - to be determined by detecting the signal emitted by the marker. Labeled RNA molecules are also used in some applications (Technical Note 4.4).

Radioactive markers are frequently used for labeling DNA molecules. Nucleotides can be synthesized in which one of the phosphorus atoms is replaced with 32P or 33P, one of the oxygen atoms in the phosphate group is replaced with 35S, or one or more of the hydrogen atoms is replaced with 3H (see Figure 1.6). Radioactive nucleotides still act as substrates for DNA polymerases and so are incorporated into a DNA molecule by any strand-synthesis reaction catalyzed by a DNA polymerase. Labeled nucleotides or individual phosphate groups can also be attached to one or both ends of a DNA molecule by the reactions catalyzed by T4 polynucleotide kinase or terminal deoxynucleotidyl transferase (Section 4.1.4). The radioactive signal can be detected by scintillation counting, but for most molecular biology applications positional information is needed, so detection is by exposure of an X-ray-sensitive film (autoradiography) or a radiation-sensitive phosphorescent screen (phosphorimaging). The choice between the various radioactive labels depends on the requirements of the procedure. High sensitivity is possible with 32P because this isotope has a high emission energy, but sensitivity is accompanied by low resolution because of signal scattering. Low-emission isotopes such as 35S or 3H give less sensitivity but greater resolution.

Health and environmental issues have meant that radioactive markers have become less popular in recent years and for many procedures they are now largely superseded by non-radioactive alternatives. The most useful of these are fluorescent markers, which are central components of techniques such as FISH (Section 5.3.2) and automated DNA sequencing (Section 6.1.1). Fluorescent labels with various emission wavelengths (i.e. of different colors) are incorporated into nucleotides or attached directly to DNA molecules, and are detected with a suitable film, by fluorescence microscopy, or with a fluorescence detector. Other types of non-radioactive labeling make use of chemiluminescent emissions, but these have the disadvantage that the signal is not generated directly by the label, but instead must be ‘developed’ by treatment of the labeled molecule with chemicals. A popular method involves labeling the DNA with the enzyme alkaline phosphatase, which is detected by applying dioxetane, which the enzyme dephosphorylates to produce the chemiluminescence.

Box Icon

Box 4.4

Techniques for studying RNA. Many of the techniques devised for studying DNA molecules can be adapted for use with RNA. Agarose gel electrophoresis of RNA is carried out after denaturation of the RNA so that the migration rate of each molecule is dependent (more...)

Image ch1f6

From: Chapter 4, Studying DNA

Cover of Genomes
Genomes. 2nd edition.
Brown TA.
Oxford: Wiley-Liss; 2002.
Copyright © 2002, Garland Science.

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.