Sequenase should be used instead of the Klenow fragment for the synthesis of oligonucleotides labeled to a high specific activity.

Sequenase should be used instead of the Klenow fragment for the synthesis of oligonucleotides labeled to a high specific activity When using oligonucleotides as probes in Southern hybridization with genomes of high complexity it is important to use very hot probes (1). This is even more necessary for genomic sequencing and in vivo footprinting using the PCR mediated linear amplification method (2, 3). For such purpose a single stranded probe or sequencing primer of high specific activity is obtained by E.coli DNA polymerase I (Klenow fragment) mediated synthesis of a complementary strand in the presence of radiolabeled nucleotides (1, 2, 3). Because separation of complementary strands of identical length is difficult and if incomplete will result in a loss of signal due to self annealing, a strategy was designed to facilitate separation of the labeled product from the unlabeled template (2; Fig. 1). A synthetic oligonucleotide is used as a template to synthesize an oligonucleotide labeled to a high specific activity by elongation of a short primer designed to leave a several nucleotide long protruding 3' end on the template. The resulting labeled extension • product was thus expected to be shorter than the template. We show here that the 3' —5' exonuclease activity of the Klenow fragment removes the protruding end during the extension reaction (Fig. 2A). This generates two oligonucleotides of the same size which most often comigrate on gel and therefore are unknowingly copurified. Replacement of Klenow fragment by an enzyme lacking 3' —5' exonuclease activity: either Sequenase™ 2.0, a modified T7 DNA polymerase (Fig. 2B), or reverse transcriptase (not shown), avoids the size reduction of the template thus allowing proper purification of the labeled extension product. Standard labeling protocol using Sequenase™: 15 picomoles* of primer and template oligonucleotides are mixed in 30 ^1 of 40 mM Tris-HCl pH 7.5, 20 mM MgCl 2 , 50 mM NaCl and the solution is incubated for 2 min. at 75°C followed by cooling to room temperature within 20 min. Then 3 /tl of 100 mM DTT, 1 fi\ of 10 mM each dTTP, dGTP, and dCTP, 20 /tl* of or> 2 P dATP (3000 Ci/mmole; 10 /iCi/^l), and 0.5 fi\ (5 u.) of Sequenase™ 2.0 (U.S.B.) are added and the sample incubated for 10 min. at room temperature. The labeled single-stranded oligonucleotide is then purified on a 15% acrylamide sequencing gel. • These amounts are suitable for a template …