An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material.
We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full- length cDNA libraries from 100 mg of total RNA. These libraries contained 8x10(5) to 8x10(6) independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5' end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.
Oh
Jung-Hwa
JH
Laboratory of Human Genomics, Division of Genomics and Proteomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-333, Korea.
Kim
Yong Sung
YS
Kim
Nam-Soon
NS
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D008970:Molecular Weight; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
D012313:RNA