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Nucleic Acids Research, 1990, Vol. 18, No. 19 5705-5711
© 1990
MOLECULAR BIOLOGY |
An ‘equalized cDNA library’ by the reassociation of short double-stranded cDNAs
Furusawa MorphoGene Project, Exploratory Research for Advanced Technology (ERATO), Research Development Corporation of Japan (JRDC) 5-9-6 Tohkohdai, Tsukuba 300-26, Japan
Received July 5, 1990. Revised September 12, 1990. Accepted September 12, 1990.
The total number of genes in higher organisms is estimated to be under one hundred thousand. However, constructing a cDNA library containing a full set of genes expressed throughout the life time of an organism, without redundancy, is a major challenge for modern biology. Towards this goal, I have tried to make a library of mouse fibroblastoid Ltk- cells with nearly equal representations of cDNA clones. Double-stranded cDNAs (ds-cDNAs) are synthesized from mRNA using an ollgo(dT)-NotI primer. After shearing to 200 – 400 bp, a synthetic linker-primer, which has one blunt and one sticky end and an internal EcoRI site, is Iigated to the cDNAs. The cDNAs are amplified by the polymerase chain reaction (PCR) using the ligated linker-primer sequence. After denaturation and reassociation of the ds-cDNAs, and isolation of single-stranded cDNAs (ss-cDNAs) by hydroxylapatite chromatography, the ss-cDNAs are again amplified by PCR. The cDNAs are digested with EcoRI and NotI, and inserted into a plasmid vector. Colony hybridization with eight probes of different abundance showed a reduction in ‘abundance variation’ from at least 20,000-fold in the original library to 40-fold in the library constructed after three cycles of equalization. This indicates the usefulness of the current procedure for making equalized cDNA libraries.
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