Minisatellites and MVR-PCR for the individual identification of parasite isolates.

In recent years, a wide variety of biochemical and molecular typing systems have been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationships among different species and subspecies. Parasite sequence-specific polymerase chain reaction (PCR)-based genotyping systems are among the most useful tools employed to date, because they can be applied to very small quantities of host-contaminated parasite material and, using repeated loci such as mini- and microsatellites, allow the identification and tracking of individual strains as well as the determination of allele and genotype frequencies in populations. Although minisatellites have been used very successfully to study parasite populations, in particular Trypanosoma brucei populations, there are some technical problems involved in the use of these markers. For example, minisatellite alleles tend to vary in a quasi-continuous fashion, making unambiguous allele identification difficult. The development of minisatellite variant repeat (MVR) mapping by the polymerase chain reaction (MVR-PCR) as a digital approach to DNA typing has overcome many of the drawbacks of minisatellite length analysis. The system assays the dispersion patterns of MVRs within minisatellite alleles, producing an easily interpretable code for each allele. This technique not only allows unequivocal allele identification but also reveals cladistic information that can be used to determine the possible genetic relationships among the different strains and subspecies. The MVR mapping technique has been applied successfully to minisatellites in the parasite Plasmodium falciparum to uniquely identify strains, and more extensively in Trypansoma brucei, where it was used to determine population structure and to examine the relationships among T. brucei subspecies, providing evidence for multiple origins of human infectively. In this chapter, the methods for genotyping of T. brucei parasites using both minisatellite allele length and MVR mapping are described in full and can be easily adapted to apply to mini-satellites in other parasites.