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Construction of an equalized cDNA library from Arabidopsis thaliana
Author(s) -
Kohchi Takayuki,
Fujishige Kotomi,
Ohyama Kanji
Publication year - 1995
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1995.08050771.x
Subject(s) - complementary dna , cdna library , primer (cosmetics) , microbiology and biotechnology , biology , genomic library , dna , plasmid , arabidopsis thaliana , insert (composites) , polymerase chain reaction , gene , genetics , chemistry , base sequence , mechanical engineering , organic chemistry , mutant , engineering
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 Not l site. A linker containing the nucleotide sequence of Sse 8387I 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.