Characterization of Size-Fractionated cDNA Libraries Generated by the in vitro Recombination-Assisted Method
| Autor: | Osamu Ohara, Gaku Mitsui, Hiroshi Kohga, Reiko Kikuno, Takahiro Nagase, Yu Takahashi, Yumiko Saga, Satoshi Kitajima, Haruhiko Koseki, Shuichi Hiraoka |
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| Rok vydání: | 2002 |
| Předmět: |
DNA
Complementary Enzyme-Linked Immunosorbent Assay Modified method Computational biology In Vitro Techniques Biology Mice Data sequences Rapid amplification of cDNA ends Complementary DNA Genetics Animals Humans Molecular Biology Gene Library Electrophoresis Agar Gel Recombination Genetic Mice Inbred BALB C Reverse Transcriptase Polymerase Chain Reaction cDNA library Size fractionated Sequence Analysis DNA General Medicine Organ Specificity Recombination In vitro recombination |
| Zdroj: | DNA Research. 9:47-57 |
| ISSN: | 1340-2838 |
| DOI: | 10.1093/dnares/9.2.47 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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