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Type: Journal article
Title: Simple, directional cDNA cloning for in situ transcript hybridization screens
Author: Tamme, R.
Mills, K.
Rainbird, B.
Nornes, S.
Lardelli, M.
Citation: Biotechniques, 2001; 31(4):938-946
Publisher: Eaton Publishing Co
Issue Date: 2001
ISSN: 0736-6205
Organisation: Centre for the Molecular Genetics of Development
Statement of
R. Tamme, K. Mills, B. Rainbird, S. Nornes, and M. Lardelli
Abstract: Here, we describe a suppression PCR-based method for directional cloning of randomly primed cDNAs from small quantities of tissue. Synthesis of the first cDNA strand is conducted on oligonucleotide-coated magnetic beads. Synthesis of the second strand is accomplished using non-specifically primed suppression PCR. This method is used to synthesize a cDNA library from zebrafish embryos at 6-9 h after fertilization. The sequencing of the clones and their use in an in situ hybridization screen to detect restricted patterns of gene transcription in zebrafish embryos showed that this method allows the rapid identification of genes that are important for development and genes that are expressed at levels unde-tectable by whole-mount in situ transcript hybridization. The random priming of cDNA alleviates the problems encountered in the identification of zebrafish genes from poly(dT)-primed cDNA clones caused by the long 3' UTRs frequently found in transcripts from this organism.
Keywords: Animals; Zebrafish; DNA, Complementary; RNA, Messenger; DNA Primers; In Situ Hybridization; Cloning, Molecular; Polymerase Chain Reaction; Sequence Analysis, DNA; Biotechnology; Transcription, Genetic; Gene Expression Regulation, Developmental; Base Sequence; Gene Library
Description: © 1983-2008 BioTechniques
RMID: 0020010620
DOI: 10.2144/01314rr05
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Appears in Collections:Molecular and Biomedical Science publications
Centre for the Molecular Genetics of Development publications

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