Desalting oligonucleotides

Dephosphorylation may be carried out using bacterial alkaline phosphomonoesterase (Heppell et al. 1962). Typical conditions are 20 fig desalted oligonucleotide mixture in 2 ml 1 M ammonium bicarbonate with 10 fig enzyme incubated for 1 hr at 37°C (De Wachter and Piers 1967). This removes 3 -terminal phosphates. Brownlee and Sanger (1967) carried out combined T1 ribonuclease and bacterial alkaline phosphatase digestion of the low molecular weight rRNA using enzyme/substrate ratios of 1/20 and 1/5 respectively in 0.01 M Tris pH 8.0 at 37°C for 1 hr. This produces fragments like XpXp...XpG where X represents any nucleoside except G. 

The resulting oligonucleotide is often of surprising purity as judged by analytic HPLC or electrophoresis, and up to 30 mg of a deoxyeicosanucleotide (20-base DNA) can be routinely obtained. Nevertheless small amounts of short sequences, resulting from capping and from base-catalysed hydrolysis, must always be removed by quick gel filtration, repeated ethanol precipitation from water (desalting), reverse-phase HPLC, gel electrophoresis, and other standard methods. 

Due to their numerous acidic hydrogens, oligonucleotides require desalting prior to MALDI analysis, e.g. by using cation exchange resins (Chap. 10.4.2). [24]... 

The NAP-25 /PD-10 column contains Sephadex G-25 and is used for a rapid desalting or buffer exchange of nucleic acids, proteins and oligonucleotides. 

Note If the experiments are highly sensitive to salt, then it is recommended to desalt the oligonucleotides by a reverse phase cartridge, such as a C-18 sep-Pak cartridge from Waters (Milford, MA), before speed-vac concentrating. Alternatively, the HPLC-purified oligonucleotides can be desalted and concentrated by ethanol precipitation. 

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). 

Larger oligonucleotide solutions can be desalted on DEAE-cellu-lose and the desalted solution concentrated by rotary evaporation, freeze drying, dialysis against Ficoll or gel filtration in a basket centrifuge depending on what apparatus is available. These methods are described below. 

Analytical Techniques and Physical Methods.— The mapping of oligonucleotides and nucleic acid digests on cellulose or cellulose-polyethyleneimine has been described recently, and columns of mercurated dextran or dihydroxyborylcellulose have been used to fractionate nucleotide mixtures. Electrophoresis on polyacrylamide gels has been advocated as a rapid method for desalting and fractionating mixtures of oligonucleotides. ... 

If oligonucleotide has been synthesized trityl-off, proceed to quench and desalting steps according to steps 8-10 in Section V.D.l. 

Load each oligonucleotide diffusion mixture onto the columns and desalt by washing with 20 mL distilled water. 

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

Quantify number of units (see Protocol 2) of all fractions including the sample load and wash. Combine only the fractions eluted with Solution B that contain large amounts of oligonucleotide. Do not combine the load and wash fractions since they are not desalted. 

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