Abstract
We tested three different standard ligation conditions (37°C for 30 min, 16°C for 24 h, and 4°C for 48 h) to generate dumbbell-shaped oligonucleotides (ODNs) as transcription factor decoys for SOX9 and αA-crystallin binding protein 1 (CRYBP1), which are positive and negative transcriptional regulators for type II collagen expression in chondrocytes. Decoy ODN for CRYBP1 was successfully produced as a “dumbbell” by all three conditions. A small amount of decoy ODN for SOX9, however, remained unligated under all three ligation conditions. Ligation at 4°C for 48 h appeared to be the least desirable for SOX9 ODN. Transfection experiments with the SOX9 ODN ligated in different conditions and a luciferase-based reporter system also supports this conclusion. In general, shorter incubation time produced more acceptable results for this ODN than incubation for a longer time. These data suggest that different ligation conditions should be tested prior to creating dumbbell-shaped ODNs for transfection experiments.
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Yamagiwa, H., Bolander, M.E. & Sarkar, G. Evaluation of different conditions for ligating dumbbell-shaped oligonucleotides. Mol Biotechnol 26, 111–115 (2004). https://doi.org/10.1385/MB:26:2:111
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DOI: https://doi.org/10.1385/MB:26:2:111