End-Labeling of DNA Fragments

Abstract

End-labeling is a rapid and sensitive method for radioactively, or nonisotopically, labeling DNA fragments and is useful where there is too little DNA to visualize by ethidium bromide staining. This can be a serious problem when separating small fragments by polyacrylamide gel electrophoresis (see Chapter 13). End-labeling has the second advantage that it can be used to produce fragments labeled at one end. Because all of the enzymes employed are specific to either the 5′ or 3′ termini of DNA molecules, it will only incorporate the label once per DNA strand. When double-stranded DNA restriction enzyme fragments are used, the label is incorporated at both ends, but can be removed from one by a second restriction enzyme digestion. This works well with DNA fragments cloned into polylinkers because one end can be removed as a tiny piece of DNA, making subsequent purification easier. These single labeled molecules can help to order restriction enzyme fragments and are a prerequisite for Maxam-Gilbert DNA sequencing (see Chapter 53). End-labeled synthetic oligonucleotides have numerous applications, such as short DNA sequence-specific probes for mutation screening (1); probes for gel retardation and Southwestern assays (2); and for sequencing PCR products where the samples are contaminated with other nucleotides (see Chapter 49).