Detection of polymorphism in the Trypanosoma cruzi TcP2 beta gene family by single strand conformational analysis (SSCA)

Gene
M P VazquezM J Levin

Abstract

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 sen...Continue Reading

References

Oct 1, 1990·Genomics·M OritaK Hayashi
Apr 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·M OritaT Sekiya
Oct 25, 1988·Nucleic Acids Research·M G LeeL H Van der Ploeg
May 1, 1995·Molecular and Biochemical Parasitology·M I CanoJ L Ramirez

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