High-performance liquid chromatography purification of 26-bp serial analysis of gene expression ditags results in higher yields, longer concatemers, and substantial time savings.
In contrast to DNA chips, serial analysis of gene expression (SAGE) is not dependent on genes having been previously identified for their monitoring. Although useful, the method can be technically challenging, and particularly the last steps including concatenation and cloning may result in less than optimal results. We propose that many of the encountered problems can be attributed to the purification of the 26-bp ditags by polyacrylamide gel electrophoresis. Low yields, gel contaminants, potential exposure to degrading enzymes during handling and lengthy separation all disfavor the method. We introduce purification of 26-bp ditags by reverse-phase high-performance liquid chromatography (HPLC) using polystyrene/divinylbenzene columns and tetraethylammonium acetate buffer with acetonitrile as mobile phase. The method is fast and gives excellent results. Ditags purified by HPLC readily ligate to high-molecular-weight concatemers leading to their efficient cloning. The method should substantially facilitate the construction of SAGE libraries.
Damgaard Nielsen
Mette
M
Bartholin Instituttet, University Hospital, Bartholinsgade 2, 1356 Copenhagen K, Denmark. mettedn@rh.dk
Millichip
Mark
M
Josefsen
Knud
K
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library
D004247:DNA