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 the small sub-unit (18S) ribosomal DNA gene from representative strains of Trypanosoma cruzi. In order to screen for sequence variation, amplification products were subsequently analysed by single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (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 differentiate 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 is the first application of PCR-linked SSCP and DGGE analysis for differentiating parasitic protozoa.
Stothard
J R
JR
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, U.K. r.stothard@lshtm.ac.uk
Frame
I A
IA
Miles
M A
MA
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D014349:Trypanosoma cruzi / Q000235:genetics*
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal