Oligonucleotide probes purification

To probe for one-dimensional diffusion, we synthesized DNA/RNA chimeric oligonucleotides. Special precautions were taken to avoid ribonuclease contamination during synthesis, purification, and use of these chimeras. For example, all water was treated with diethylpyrocarbonate before exposure to the chimeras. Ribonucleotide 2 -hydroxyl groups were deprotected with 1 M tetrabutyl ammonium fluoride in dimethyl formamide (Aldrich Chemical Milwaukee, WI). Purified oligonucleotides were labeled on the 5 end with [y-32p]ATP (duPont Wilmington, DE) by T4 kinase (Promega Madison, WI), and desalted with a Nick gel filtration column (Pharmacia Uppsala, Sweden). 

There are, however, potential disadvantages to the use of photophysical probes in detecting sequence-directed DNA structure and dynamics. The probe molecule may require complicated synthesis or tedious incorporation into the DNA with subsequent purification the probe may introduce structural or dynamic distortions into the DNA. The first point has been well addressed for commonly used fluorescent probes such as fluorescein and tetramethylrhodamine, which are commercially available, and even oligonucleotides bearing such molecules in defined positions are commercially available. (It is also worth mentioning that some of the most informative NMR experiments rely on isotopic substitutions at the base or sugar, a challenging synthetic task as well.)... 

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