Towards the development of an RT-PCR protocol for malaria parasite genes

Abstract

Malaria is a life-threatening parasitic disease, which is becoming increasingly problematic in Sub-Saharan Africa due to the spread of drug-resistant parasite strains. Novel therapeutic targets and drugs are thus required to successfully treat and/or prevent malaria. The aim of this study was the identification and characterisation of an amino acid transporter gene of the human malaria parasite, Plasmodium falciparum, as a possible therapeutic target. Since no sequence information was available, 3 '-RACE (a modified form of RT-PCR) was chosen as a method to obtain the gene sequence. Some of the obstacles encountered and the way that they were addressed are described. The synthesis of a representative, full-length, uncloned cDNA library of the AT-rich malaria genome was one focal point of the study. The best quality cDNA was obtained when total RNA was used, with a 'hot start' protocol prior to synthesis. Three gene specific primers were designed, based on an identified consensus sequence of multiply aligned amino acid permeases from other organisms. The sequence of a transporter gene was not obtained (due mostly to mispriming), though many partial gene sequences of known therapeutic targets were obtained. Numerous rearrangements of the inserts by the bacterial host were observed and methods of minimising this were suggested. The absence of the amino acid transporter may be due to a template concentration that is too low, a slightly different consensus sequence or the absence of the corresponding gene in the malaria parasite. Possible means of continuing with further studies are proposed. A partial sequence of one novel protein, of the large (60S) ribosomal subunit was obtained. It was confirmed, by hybridisation of a probe to total RNA, that the sequence was of malarial origin.