Polymerase chain reaction-based construction of cDNA libraries from minute amounts of third-stage and fourth- and fifth-stage larvae of Dictyocaulus viviparus.
A strategy is described for the amplification and cloning of cDNA from minute amounts of Dictyocaulus viviparus larvae. Initially, third-stage larvae (L3) were used to establish the procedure. Amplification of cDNA synthesized from approximately 400 ng total RNA from 5,000 L3 generated products that were more than 800 bp in length. The unidirectional cloning of amplified cDNA products led to the construction of a UNI ZAP c DNA library with 1 x 10(6) clones. Screening with a homologous oligo(dT)-primed digoxigenin-labeled cDNA probe as well as sequencing of seven randomly picked clones confirmed the successful cloning of lung-worm cDNA. Subsequently, approximately 600 ng total RNA was isolated and polymerase chain reaction (PCR) products of up to 2,400 bp were amplitied from 400 fourth- and fifth-stage larvae (L4/L5). Cloning of these products resulted in a L4/L5 cDNA library of D. viviparus consisting of 5 x 10(5) recombinant clones. In all, 11 clones were randomly picked and sequenced, all revealing typical mRNA/cDNA characteristics. Comparison of the predicted amino acid sequence of the 5' end of clone DvL5/7 revealed 100% homology with the actin gene of several other helminths.
von Samson-Himmelstjerna
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Institut für Parasitologie, Tierärztliche Hochschule Hannover, Germany.
Wunderlich
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Mühlschlegel
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Frosch
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Schnieder
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D000199:Actins / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004021:Dictyocaulus / Q000235:genetics* / Q000254:growth & development; D003576:Electron Transport Complex IV / Q000235:genetics; D005243:Feces / Q000469:parasitology; D015723:Gene Library; D007814:Larva / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018510:RNA, Helminth / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000199:Actins; D018076:DNA, Complementary; D018510:RNA, Helminth; D003576:Electron Transport Complex IV