ABSTRACT
Contents Molecular biology studies usually involve molecular cloning, characterization, and analysis of gene expression. These procedures rely heavily on nucleic acid hybridization, such as Southern blots, Northern blots, and dot blots. One of the most important aspects of these protocols is the use of either radioactively or nonradioactively labeled nucleic acids (deoxyribonucleic acid [DNA] or ribonucleic acid [RNA]) as probes that specifically hybridize with their complementary DNA or RNA strands. The quality of the probe plays an essential role in detecting specific DNA or RNA sequences of interest. Therefore, the preparation of a probe with high specific activity is critical in nucleic acid hybridization.1 The present chapter describes in detail reliable methods for the labeling of DNA and RNA.2-5 These methods are well established and have been routinely used in our laboratory. 0-8493-0815-1/04/$0.00+$ 1.50
I. Radioactive Labeling Methods
A. Nick-Translation Labeling of dsDNA
The principle of nick translation is that one strand of a double-strand DNA (dsDNA) molecule is nicked with DNase I, generating free 3′-hydroxyl ends within the unlabeled DNA. E. coli DNA polymerase I, by virtue of its 5′ to 3′ exonucleolytic activity, removes nucleotides from the 5′ side of the nick and simultaneously adds new nucleotides to the 3′-hydroxyl terminus of the nick. During the incorporation of new nucleotides, one of four deoxyribonucleotides is radioactively labeled (e.g., [α-32P]dATP or [α-32P]dCTP, commercially available) and is incorporated into the new strand by a base complementary to the template. In this way, a high specific activity (108 cpm/µg) of labeled DNA can be obtained using [α-32P]dATP or [α-32P]dCTP. The protocol given below works well in our hands.
