Toward a whole cDNA catalog: construction of an equalized cDNA library from mouse embryos.
The construction of a cDNA library containing all genes without redundancy is one of the major technical challenges for biology. Toward this goal, we have developed an equalized (normalized) cDNA library from mRNA pools derived from mouse embryos that cover the entire period of mouse ontogenesis. Colony hybridization analyses with 11 genes showed the reduction of abundance variation from at least 6000-fold in the unequalized S-library to approximately 33-fold in the EIII-library, which was constructed after three cycles of equalization procedure. Limiting dilution PCR analyses with 26 tissue-specific genes showed the reduction of abundance variation from at least approximately 1,000,000-fold in the S-cDNA mixture to approximately 100-fold in the EIII-cDNA mixture. Based on these analyses, we estimate that at least 15,000 independent cDNA clones are included with little redundancy in the EIII-cDNA library. This will be a useful resource for mouse biology as well as the mouse genome project.
Takahashi
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Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48202.
Ko
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MS
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D016678:Genome; D051379:Mice / Q000196:embryology / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger