Two-dimensional gene expression fingerprinting

In the present study we have developed the two-dimensional Gene Expression Fingerprinting (2-D CEF) procedure suitable for gene expression analysis an d new gene discovery. The procedure is based on the two-dimensional gel dis play of 3'-terminal cDNA restriction fragments produced by one primary (fir st dimension) and several sequential secondary restriction digestions, Many thousands of individual sequences per cDNA sample can be visualized using this approach, which is also characterized by a high reproducibility, predi ctable spatial location of cDNA fragments on 2-D gels, and the potential fo r identifying cDNA fragments solely on the basis of their two-dimensional c oordinates, Using this 2-D GEF method, we analyzed and compared the gene ex pression patterns of two related primitive hematopoietic cell lines, Kg-1 a nd Kg-1a. A total of 25 candidate differentially expressed sequences were i dentified, and for 75% of them the presumed expression pattern was confirme d by Northern blotting or reverse transcription-polymerase chain reaction. We also demonstrated that for 70% of bands, correct prediction of their ide ntity could be made on the basis of two-dimensional coordinates, whereas th e major part of incorrect predictions was caused by insufficient database q uality. (C) 2001 Academic Press.