A nucleotide-constrained single base extension method for improved detection of minority alleles in Plasmodium

In genetically mixed Plasmodium infections, minority alleles may have a significant role in drug resistance and other responses to selective pressures. Many available methods to detect single nucleotide polymorphisms representing minority alleles either are not sensitive enough to detect these rare alleles or are limited in the number of loci to be screened in a single reaction. In order to achieve highly sensitive, multiplex SNP genotyping, we have developed a nucleotide-constrained method based on the traditional single base extension approach. Here, we report results when using standard and nucleotide-constrained reactions to determine alleles at nine SNP loci in the trap gene of Plasmodium relictum. This innovative method offers great improvements in detection limits while maintaining the accuracy and multiplex capabilities of single base extension SNP genotyping.