Construction of an equalized cDNA library from Arabidopsis thaliana.
Using a kinetic approach, a cDNA library composed of almost equal representations of all genes expressed in the aerial parts of 2-week-old Arabidopsis was constructed. A cDNA was synthesized with an oligo dT primer containing a Notl site. A linker containing the nucleotide sequence of Sse8387I which recognizes octanucleotides was added at the ends of the synthesized cDNA. The cDNA was amplified by the polymerase chain reaction (PCR), denatured, and reassociated under modified conditions. Thereafter, the remaining single-stranded DNA was converted to double-stranded DNA and amplified by PCR. These equalization steps were repeated three times and the products were cloned unidirectionally into a plasmid vector. Equalization was evaluated by colony hybridization and DNA sequencing. This approach will be applicable to construct a cDNA library suitable for subtraction, differential screening, and expression screening, especially for mRNA species present at very low concentrations in a few cells of a specific tissue.
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan.
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Kohchi
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Fujishige
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Ohyama
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D005069:Evaluation Studies as Topic; D015723:Gene Library; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary