Exp Mol Med.  2003 Dec;35(6):586-590.

An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material

Affiliations
  • 1Laboratory of Human Genomics, Division of Genomics and Proteomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-333, Korea. nskim37@kribb.re.kr

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.

Keyword

cDNA library; full-length; oligo-capping; total RNA

MeSH Terms

Base Sequence
Cloning, Molecular/*methods
*Gene Library
Molecular Weight
RNA/*chemistry/genetics/*isolation & purification
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