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Use of activated charcoal for adsorption and elution of ribooligonucleotides

✍ Scribed by Stanley Mandeles; Harold O. Kammen

Publisher
Elsevier Science
Year
1966
Tongue
English
Weight
280 KB
Volume
17
Category
Article
ISSN
0003-2697

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✦ Synopsis

The purification of oligonucleotides for base sequence analysis often requires the separation of these materials from concentrated salt solutions, or from 7 M urea. This separation is commonly carried out by adsorption of dilute oligonucleotide solutions on columns of DEAEcellulose or DEAE-Sephadex, elution with NH,HCO,, and removal of the water and NH,HCO, in Z~UCUO (1). This procedure is tedious and time consuming and is conveniently applied only to tetramers and shorter oligomers.

Activated charcoal has been successfully employed for the desalting and recovery of mononucleotides

(2). Because of its adaptability to rapid filtration techniques, we have examined the usefulness of charcoal adsorption and elution for the isolation of oligonucleotides.

Ribomononucleotides, both the 2',3'-and the 5'-isomers, were purchased from P-L Biochemicals, Inc., polyadenylic acid from Miles Laboratories, and T,-ribonuclease from Sankyo Co., Ltd., Tokyo. Tobacco mosaic virus (TMV) RNA was prepared by a variation (3) of previously described methods (4, 5).

T,-ribonuclease digests of TMV RNA were chromatographed on DEAE-Sephadex to give oligonucleotide fractions which differed on the basis of chain length (6). The individual dimers and trimers were resolved from these DEAE-Sephadex fractions by subsequent chromatography on Dowex-1 chloride (x2), using a NaCl-HCl gradient (7). Oligoadenylic acids were prepared by alkaline hydrolysis is of polyadenylic acid, followed by chromatography on DEAE-Sephadex, as described above.

Acid-washed activated charcoal, Norit A, was obtained from Pfanstiehl Laboratories, Inc., Waukegan, Illinois, and was suspended at a concentration of 100 mg/ml in 0.001 M phosphate-O.001 M pyrophosphate buffer, pH 6.0. For desalting, the chilled nucleotide solutions were adjusted to a pH of about 3 and the Norit A suspension added, 5 mg of

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