Vector-capping: a simple method for preparing a high-quality full-length cDNA library.
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Kato S, Ohtoko K, Ohtake H, Kimura T
Vector-capping: a simple method for preparing a high-quality full-length cDNA library.
DNA Res. 2005 Feb 28;12(1):53-62.
- PubMed ID
- 16106752 [ View in PubMed]
- Abstract
Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.