An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material.
- Author:
Jung Hwa OH
1
;
Yong Sung KIM
;
Nam Soon KIM
Author Information
1. Laboratory of Human Genomics, Division of Genomics and Proteomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-333, Korea. nskim37@kribb.re.kr
- Publication Type:Case Report ; Research Support, Non-U.S. Gov't
- Keywords:
cDNA library;
full-length;
oligo-capping;
total RNA
- MeSH:
Base Sequence;
Cloning, Molecular/*methods;
*Gene Library;
Molecular Weight;
RNA/*chemistry/genetics/*isolation & purification
- From:Experimental & Molecular Medicine
2003;35(6):586-590
- CountryRepublic of Korea
- Language:English
-
Abstract:
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 microg 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.
