Two-dimensional gene expression fingerprinting.
In the present study we have developed the two-dimensional Gene Expression Fingerprinting (2-D GEF) procedure suitable for gene expression analysis and new gene discovery. The procedure is based on the two-dimensional gel display of 3'-terminal cDNA restriction fragments produced by one primary (first 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, predictable spatial location of cDNA fragments on 2-D gels, and the potential for identifying cDNA fragments solely on the basis of their two-dimensional coordinates. Using this 2-D GEF method, we analyzed and compared the gene expression patterns of two related primitive hematopoietic cell lines, Kg-1 and Kg-1a. A total of 25 candidate differentially expressed sequences were identified, and for 75% of them the presumed expression pattern was confirmed by Northern blotting or reverse transcription-polymerase chain reaction. We also demonstrated that for 70% of bands, correct prediction of their identity could be made on the basis of two-dimensional coordinates, whereas the major part of incorrect predictions was caused by insufficient database quality.
Shmelkov
S V
SV
New York Blood Center, 310 East 67th Street, New York, New York 10021, USA.
Visser
J W
JW
Belyavsky
A V
AV
D015152:Blotting, Northern; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D006412:Hematopoietic Stem Cells; D012333:RNA, Messenger; D015183:Restriction Mapping / Q000379:methods
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger