Efficient 5'-end labeling of oligonucleotides containing self-complementary sequences.

Synthetic oligonucleotides have become basic tools in molecular biology research and are commonly used for cloning and characterization of genes, analysis of gene expression and so on. Frequently, the success of techniques such as DNA and RNA sequencing, screening of libraries, nuclease S1 and RNase protection assays or polymerase chain reaction (PCR) amplification depends on appropriate design and labeling of the oligonucleotides used. At the time of oligonucleotide design, sequences with the potential to self-anneal should be avoided because self-complementarity of oligonucleotides is a factor that directly interferes with sequencing, hybridization and labeling processes. However, when oligonucleotides are designed for cloning purposes, they often include endonuclease restriction sites (e.g., palindromic sequences) to simplify the cloning process. We report a method that allows the labeling of oligonucleotides even though they contain strong secondary structures. In the development of the technique, we took advantage of previous reports indicating that dimethyl sulfoxide (DMSO) reduces interand intra-strand annealing (4) and improves sequencing and PCRs (5). It has been shown that DMSO has its primary effect on melting temperature of primers (1). Thus, we assayed the effect of this denaturing agent on the labeling efficiency of oligonucleotides with the potential to form stable secondary structures. The oligonucleotide 70 II, shown in Figure 1, was chosen because of repeated failures to label it using the standard T4 polynucleotide kinase method (3). Prior to labeling, the oligonucleotide was incubated for 2 min at 70°C in the presence of 5% DMSO. Immediately, the tube was chilled on ice, and 1 μL (25 ng) of the oligonucleotide solution was added to the labeling reaction mixture (50 mM TrisHCl, 10 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol, 0.1 mM spermidine, pH 8.2), containing 50 μCi of [γ32P]ATP (3000 Ci/mmol; Amersham International plc, Bucks, England, UK) and 10 U of T4 polynucleotide kinase (Boehringer Mannheim GmbH, Mannheim, Germany) in a final volume of 10 μL. The mixture was incubated at 37°C for 30 min. Afterwards, the reaction was stopped by the addition of 5 μL of 500 mM EDTA (pH 8.0), and the volume was adjusted to 100 μL with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The labeled oligonucleotide was purified using a Sephadex G-50 Column (Pharmacia Biotech, Piscataway, NJ, USA), and the incorporated cpm were measured in a scintillation counter. In parallel, as a control, the same oligonucleotide was labeled using the standard technique without the DMSO treatment (3). Also, according to a suggestion from Oligos Etc. (Wilsonville, OR, USA), a labeling reaction was performed in the presence of 2× T4 polynucleotide kinase buffer (Boehringer Mannheim GmbH). This latter procedure was found to solve the labeling problems of some, but not all, oligonucleotides (unpublished). Table 1 summarizes the labeling efficiency values obtained by the different methods. The results indicate that denaturation of oligonucleotide 70 II in the presence of DMSO increased 20-fold the 32P incorporation efficiency relative to standard methods. The high specific activity of the labeled, DMSO-treated oligonucleotide, 8.4 × 108 cpm/μg, should be noted. The efficient labeling of oligonu-