Vector-capping: a simple method for preparing a high-quality full-length cDNA library.
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.
Kato
Seishi
S
Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan. seishi@rehab.go.jp
Ohtoko
Kuniyo
K
Ohtake
Hideki
H
Kimura
Tomoko
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009711:Nucleotides; D012315:RNA Caps; D012333:RNA, Messenger