title: Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method.
abstract: We here modified a previously reported method for the construction of cDNA libraries by employing an in vitro recombination reaction to make it more suitable for comprehensive cDNA analysis. For the evaluation of the modified method, sets of size-selected cDNA libraries of four different mouse tissues and human brain were constructed and characterized. Clustering analysis of the 3' end sequence data of the mouse cDNA libraries indicated that each of the size-fractionated libraries was complex enough for comprehensive cDNA analysis and that the occurrence rates of unidentified cDNAs varied considerably depending on their size and on the tissue source. In addition, the end sequence data of human brain cDNAs thus generated showed that this method decreased the occurrence rates of chimeric clones by more than fivefold compared to conventional ligation-assisted methods when the cDNAs were larger than 5 kb. To further evaluate this method, we entirely sequenced 13 human unidentified cDNAs, named KIAA1990-KIAA2002, and characterized them in terms of the predicted protein sequences and their expression profiles. Taking all these results together, we here conclude that this new method for the construction of size-fractionated cDNA libraries makes it possible to analyze cDNAs efficiently and comprehensively.
authors.affiliation: Department of Human Gene Research, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan. ohara@kazusa.or.jp
authors.forename: Osamu
authors.initials: O
authors.lastname: Ohara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagase
authors.forename: Gaku
authors.initials: G
authors.lastname: Mitsui
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kohga
authors.forename: Reiko
authors.initials: R
authors.lastname: Kikuno
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hiraoka
authors.forename: Yu
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kitajima
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Saga
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Koseki
mesh_terms: D000818:Animals; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D009928:Organ Specificity; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary