Simple, directional cDNA cloning for in situ transcript hybridization screens.
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 nonspecifically 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 undetectable 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.
Tamme
R
R
The University of Adelaide, Australia.
Mills
K
K
Rainbird
B
B
Nornes
S
S
Lardelli
M
M
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D015027:Zebrafish / Q000196:embryology / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger