In this study, we present a P. falciparum
ORF-specific microarray utilizing 70 mer oligonucleotides as individual microarray elements. This approach helped to overcome potential problems originating from low PCR amplification and allowed us to select probes with a high specificity, thereby minimizing potential cross-hybridization. Moreover, the oligonucleotide-selection algorithm allowed a balanced GC content (around 28%) across the entire microarray set, which is significantly higher than the plasmodial genome average, which is 19.4% with 23.7% in coding regions [4
Application of the ArrayOligoSelector is not restricted to the P. falciparum genome, but is broadly useful for the automated selection of hybridization probes for a range of species. The flexibility of the selection parameters controlling stringency of uniqueness, self-binding, complexity, user-defined filters and GC content, allows the selection of oligonucleotides appropriate for any genome.
Evaluation of results from derivative control oligonucleotides showed that long oligonucleotides could tolerate 10% mismatches; however, alteration of the target sequence by more then 20% eliminated most of the hybridization signal. Therefore, small sequencing errors and natural variation among isolates are not likely to impact on sensitivity. These performance characteristics imply that the array design for this effort can accommodate the study of essentially any P. falciparum strain with a high degree of specificity.
At present, the P. falciparum
microarray used in this study consists of approximately 6,000 gene-specific elements corresponding to the majority of the total coding content predicted for the P. falciparum
genome. As new sequence and improved gene predictions arise, additional elements will be added to this evolving platform. Moreover, the present oligonucleotide representation could be further extended for investigation of several unusual P. falciparum
genetic and transcriptional phenomena, including antisense mRNA transcription [48
] and alternative splicing and/or transcriptional initiation [49
]. This may be achieved by designing exon-specific array features, as well as antisense oligonucleotides. The oligonucleotide collection could also be expanded by sequences corresponding to intergenic genomic regions. Inclusion of such elements was found to be extremely useful for identifying protein-binding DNA regions by chromatin-immunoprecipitation as well as genes not detected by automated gene-prediction algorithms [51
Within both the trophozoite and schizont categories, large numbers of genes belong to functionally related processes. These include genes encoding ribosomal subunits, multiple factors for transcription and translation, enzymes of biosynthetic and catabolic pathways, or merozoite adherence and invasion machinery. These results are consistent with predictions that a large number of plasmodial genes undergo strict stage-specific transcriptional regulation, and that such (co-)regulation is shared among functionally related genes [15
]. Naturally, a 'fine-resolution' global gene-expression profile including the different steps of the plasmodial life cycle for multiple divergent strains will be necessary to characterize fully the intraerythrocytic life of the parasite. At present, our laboratory is analyzing a global gene-expression profile of the 48-hour erythrocytic life cycle with 1-hour resolution for three strains of P. falciparum.
In a number of model organisms, high-resolution gene-expression maps have served as extremely powerful tools for discovery and characterization of novel genes as well as exploration of multiple cellular functions [9
]. The gene-expression maps typically comprise genome-wide expression profiles at a number of different stages of cellular development, profiles of multiple strains and genetic variants, and global expression responses to number of growth perturbations and growth-inhibitory drugs. Following a similar approach in P. falciparum
is most likely to provide substantial information about the many ORFs that lack functional annotation. Further understanding of cellular physiology of this parasite including basic metabolic functions and the intricate interactions between the parasite cell and human host immune system will be a key step in uncovering new targets for antimalarial drug discoveries and vaccine development.