Preparation of full-length cDNA libraries: focus on metazoans.
Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.
Harada
Masako
M
Genome Exploration Research Group, Genomic Sciences Center (GSC), RIKEN, Yokohama, Japan.
Hayashizaki
Yoshihide
Y
D000818:Animals; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D020224:Expressed Sequence Tags; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA