An 'equalized cDNA library' by the reassociation of short double-stranded cDNAs

Research output: Contribution to journalArticle

119 Citations (Scopus)

Abstract

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 oligo(dT)-Notl 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 ligated 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 Notl, 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.

Original languageEnglish
Pages (from-to)5705-5711
Number of pages7
JournalNucleic Acids Research
Volume18
Issue number19
Publication statusPublished - 1990 Oct 11
Externally publishedYes

Fingerprint

CDNA
Gene Library
Complementary DNA
Polymerase chain reaction
Libraries
Genes
Denaturation
Polymerase Chain Reaction
Chromatography
Shearing
Redundancy
Fold
cDNA
Gene
Durapatite
Equalization
Plasmids
Clone
Clone Cells
Messenger RNA

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

An 'equalized cDNA library' by the reassociation of short double-stranded cDNAs. / Ko, Minoru.

In: Nucleic Acids Research, Vol. 18, No. 19, 11.10.1990, p. 5705-5711.

Research output: Contribution to journalArticle

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