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Preparation of full-length cDNA libraries: focus on metazoans
Ist Teil von
Methods in molecular biology (Clifton, N.J.), 2009, Vol.533, p.69
Ort / Verlag
United States
Erscheinungsjahr
2009
Quelle
MEDLINE
Beschreibungen/Notizen
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.