POLYMERASE CHAIN REACTION-BASED CONSTRUCTION OF CDNA LIBRARIES FROM MINUTE AMOUNTS OF 3RD-STAGE AND 4TH-STAGE AND 5TH-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-sta ge larvae (L3) were used to establish the procedure. Amplification of cDNA synthesized from approximately 400 ng total RNA from 5,000 L3 gen erated products that were more than 800 bp in length. The unidirection al cloning of amplified cDNA products led to the construction of a UNI ZAP cDNA library with 1 X 10(6) clones. Screening with a homologous o ligo(dT)-primed digoxigenin-labeled cDNA probe as well as sequencing o f seven randomly picked clones confirmed the successful cloning of lun gworm cDNA. Subsequently, approximately 600 ng total RNA was isolated and polymerase chain reaction (PCR) products of up to 2,400 bp were am plitied from 400 fourth- and fifth-stage larvae (L4/L5). Cloning of th ese products resulted in a L4/L5 cDNA library of D. viviparus consisti ng of 5 X 10(5) recombinant clones. In all, 11 clones were randomly pi cked and sequenced, all revealing typical mRNA/cDNA characteristics. C omparison of the predicted amino acid sequence of the 5' end of clone DvL5/7 revealed 100% homology with the actin gene of several other hel minths.