Reverse Transcription-Polymerase Chain Reaction Construction of Plasmid-based, Full-length cDNA Libraries from Leishmania infantum for in Vitro Expression Screening

Abstract

We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5′ and 3′ ends of transspliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen). We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.