Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

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As an alternative to 5- and 3-end-labeling, probes of high specific activities can be obtained using the Klenow fragment of E. coli DNA Pol I to synthesize a strand of DNA complementary to the synthetic oligonucleotide (Studencki and Wallace 1984; Ullrich et al. 1984b; for review, see Wetmur 1991). In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions (see Fig. 1). With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 1010 cpm/g of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated (see below), this method is generally not used to prepare nonradiolabeled oligonucleotides. To obtain the best results, consider the points discussed in the box Optimizing Oligonucleotide Labeling Using the Klenow Fragment.

OPTIMIZING OLIGONUCLEOTIDE LABELING USING THE KLENOW FRAGMENT


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