Detection of polymorphism in the Trypanosoma cruzi TcP2 beta gene family by single strand conformational analysis (SSCA).
Single strand conformation analysis (SSCA) is a technique that has been used to detect point mutations. We explored its usefulness in the analysis of four different members of the Trypanosoma cruzi TcP2 beta gene family and its suitability for detection of polymorphism in different parasite strains. The availability of primers covering a 97-bp sequence at the 5' end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic conditions: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4 degrees C. Shifts in mobility were radically dependent on the migration condition. Both 97-bp and 321-bp amplicons were best resolved at 4 degrees C, without glycerol. Amplification products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the cloned genes. The results herein demonstrate the usefulness of SSCA to differentiate forms of a complex protozoan gene family, and to scan its polymorphic nature. Furthermore, due to the remarkable sensitivity of the technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project.
Vázquez
M P
MP
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires, Argentina.
Beldjord
C
C
Lorenzi
H
H
Bienvenu
T
T
Levín
M J
MJ
D000818:Animals; D001483:Base Sequence; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D005810:Multigene Family; D010750:Phosphoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015800:Protozoan Proteins / Q000235:genetics*; D012269:Ribosomal Proteins; D016324:Sequence Tagged Sites; D014349:Trypanosoma cruzi / Q000235:genetics*
D010750:Phosphoproteins; D015800:Protozoan Proteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal