High-efficiency full-length cDNA cloning by biotinylated CAP trapper.
We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.
Carninci
P
P
Genome Science Laboratory, Tsukuba Life Science Center, Ibaraki, Japan.
Kvam
C
C
Kitamura
A
A
Ohsumi
T
T
Okazaki
Y
Y
Itoh
M
M
Kamiya
M
M
Shibata
K
K
Sasaki
N
N
Izawa
M
M
Muramatsu
M
M
Hayashizaki
Y
Y
Schneider
C
C
D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics; D012315:RNA Caps / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D019809:Streptavidin
D001426:Bacterial Proteins; D018076:DNA, Complementary; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D001710:Biotin; D019809:Streptavidin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012259:Ribonuclease, Pancreatic