Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns.
We have analyzed an ordered library of 4,608 cDNA clones from the CEM human leukemic cell line. The aim was to facilitate gene retrieval, to enable immediate access to cDNA clones and to provide information on the protein expression of the individual clones in a 2D gel readout. The matrix array of 24 x 16 x 12, each position of which contained lambda jacII phage from one plaque, enabled us to establish pools of clones along the three axes (24 pools of complexity 192 cloned entities, 16 pools of complexity 288 and 12 pools of complexity 384). The total cDNA complexity is here reduced to such a level that spots which in more complex gels served as landmark spots are not present in each pool, and thus cannot serve as landmarks anymore. The image analysis of such gels and especially the matching of spots is not reliable under these circumstances. In order to achieve reliable matching, additional samples were created, such that pools were co-electrophoresed according to a special concatenation scheme; these samples then contained over-lapping elements (e.g., pools 1 + 2 + 3 and 3 + 4 + 5 have at least those spots in common, which originate from pool 3). This approach turned out to be feasible and we have completed the matching of one half of the ordered library. Already from the present stage of analysis we have obtained valuable information on the cDNA library and on the distribution of clones in this library.
Basel Institute for Immunology, Switzerland.
I
I
Lefkovits
J R
JR
Frey
L
L
Kuhn
J R
JR
Kettman
G
G
Béhar
C
C
Auffray
J P
JP
Hoffmann
C
C
Coleclough
D018076:DNA, Complementary / Q000097:blood*; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D015698:Genomic Library; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007938:Leukemia / Q000097:blood / Q000235:genetics*; D008214:Lymphocytes / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary