High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper |
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Authors: | Carninci Piero; Westover Arthur; Nishiyama Yoko; Ohsumi Tomoya; Itoh Masayoshi; Nagaoka Sumiharu; Sasaki Nobuya; Okazaki Yasushi; Muramatsu Masami; Schneider Claudio; Hayashizaki Yoshihide |
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Institution: | 1Genome Science Laboratory, Tsukuba Life Science Centre, The Institute of Physical and Chemical Research (RIKEN) 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan
2Tsukuba University Medical School 1-1-1, Tennodai, Tsukuba, Ibaraki 305, Japan
3Laboratorio Nazionale CIB Area Science Park, 99 Padriciano, 34012 Trieste, Italy |
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Abstract: | We report here an improved protocol for the preparation of full-lengthcDNA libraries that improves the previously reported method(Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics,137, 327336), that allows long cDNAs to be cloned moreefficiently. One potential disadvantage of the original biotinylatedCAP trapper protocol is the exposure of mRNA to chemical andenzymatic attacks during the biotinylation of the cap structure,before the first-strand cDNA synthesis (and selection of full-lengthcDNA by biotinylated cap). Here, we show that the biotinylationof the cap structure is very specific and effective even ifbiotinylation is performed on the mRNA/cDNA hybrid producedby the first-strand cDNA synthesis reaction. Consequently, mRNAremains protected from chemical and enzymatic degradation duringthe overnight biotinylation step, thus making it possible toselect full-length cDNAs of longer average size. We herein reportthe efficiency and specificity of the new version of the protocolfor cap structure biotinylation and capture of full-length cDNA. |
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Keywords: | full-length cDNA biotin technology cDNA library |
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