USE OF POLYMERASE CHAIN REACTION-BASED SINGLE-STRAND CONFORMATIONAL POLYMORPHISM AND DENATURING GRADIENT GEL-ELECTROPHORESIS METHODS FOR DETECTION OF SEQUENCE VARIATION OF RIBOSOMAL DNA OF TRYPANOSOMA-CRUZI

Polymerase chain reaction (PCR) was used to amplify the V1 region of t he small sub-unit (18S) ribosomal DNA gene from representative strains of Trypanosoma cruzi. In order to screen for sequence variation, ampl ification products were subsequently analysed by single strand conform ational polymorphism (SSCP) and denaturing gradient gel electrophoresi s (DGGE) techniques. SSCP could not detect sequence variation within T . cruzi, although electrophoretic profiles were clearly distinct from both Leishmania donovani and Leishmania braziliensis. DGGE could diffe rentiate strains of T. cruzi and it appears that there are at least 2 18S V1 sequences of this multi-gene family within each strain examined , contrasting with Leishmania spp. where only 1 was identified. This i s the first application of PCR-linked SSCP and DGGE analysis for diffe rentiating parasitic protozoa. (C) 1997 Australian Society for Parasit ology.