Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension

Abstract

In this procedure, synthesis of cDNA is performed in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP. After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of Escherichia coli DNA Pol I. Because the concentrations of dNTP in the first reaction are nonlimiting, both the amounts and size of cDNA generated are greater than those achieved in standard labeling protocols. The subtractive hybridization step can therefore be performed with higher efficiency. Because the resulting population of cDNA is not vulnerable to radiolytic cleavage, it can be stored indefinitely and radiolabeled to higher specific activity when needed. The protocol works best when the cDNA synthesized in the initial synthetic reaction is full length or close to it. For this reason, synthesis of cDNAs is primed by oligo(dT) rather than random hexanucleotide primers. In contrast, the subsequent radiolabeling reaction is primed by random oligonucleotides, yielding shorter DNA products whose size is ideal for hybridization.