Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening.
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 trans-spliced 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.
Couvreur
Bernard
B
Laboratory of Applied Genetics, IBMM, Free University of Brussels, Gosselies, Belgium.
Bollen
Alex
A
Le Ray
Dominique
D
Dujardin
Jean-Claude
JC
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D015723:Gene Library; D018314:Leishmania infantum / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D016054:DNA, Protozoan; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016053:RNA, Protozoan