High efficiency selection of full-length cDNA by improved biotinylated cap trapper.
We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.
Carninci
P
P
Genome Science Laboratory, Tsukuba Life Science Centre, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Westover
A
A
Nishiyama
Y
Y
Ohsumi
T
T
Itoh
M
M
Nagaoka
S
S
Sasaki
N
N
Okazaki
Y
Y
Muramatsu
M
M
Schneider
C
C
Hayashizaki
Y
Y
D000818:Animals; D001710:Biotin / Q000737:chemistry*; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D017403:In Situ Hybridization; D051379:Mice; D008815:Mice, Inbred Strains; D011755:Pyrophosphatases / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D012315:RNA Caps / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000378:metabolism
D018076:DNA, Complementary; D012315:RNA Caps; D012333:RNA, Messenger; D012313:RNA; D001710:Biotin; D012259:Ribonuclease, Pancreatic; D011755:Pyrophosphatases