Rahul Mitra

Human-blind probes and primers for dengue virus identification: Exhaustive analysis of subsequences present in the human and 83 dengue genome sequences

Reliable detection and identification of pathogens in complex biological samples, in the presence of contaminating DNA from a variety of sources, is an important and challenging diagnostic problem for the development of field tests. The problem is compounded by the difficulty of finding a single, unique genomic sequence that is present simultaneously in all genomes of a species of closely related pathogens and absent in the genomes of the host or the organisms that contribute to the sample background. Here we describe ‘host‐blind probe design’– a novel strategy of designing probes based on highly frequent genomic signatures found in the pathogen genomes of interest but absent from the host genome. Upon hybridization, an array of such informative probes will produce a unique pattern that is a genetic fingerprint for each pathogen strain. This multiprobe approach was applied to 83 dengue virus genome sequences, available in public databases, to design and perform in silico microarray experiments. The resulting patterns allow one to unequivocally distinguish the four major serotypes, and within each serotype to identify the most similar strain among those that have been completely sequenced. In an environment where dengue is indigenous, this would allow investigators to determine if a particular isolate belongs to an ongoing outbreak or is a previously circulating version. Using our probe set, the probability that misdiagnosis at the serotype level would occur is ≈u200a1u2003:u200310150.

Ultraspecific probes for high throughput HLA typing

BackgroundThe variations within an individuals HLA (Human Leukocyte Antigen) genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies.ResultsWe present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are ultraspecific for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus.ConclusionThe assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.

High throughput drug screening by direct RNA profiling on DNA sensors

Abstract The feasibility of DNA microarray sensor technology as a routine technique of molecular pharmacology to perform high throughput drug screening and the advantages of directly labeled RNA for a high throughput experiment are presented in this paper. A novel, single-step direct chemical labeling method for DNA microarray target samples has been developed to reduce the sample amount, cost, time and error of the experiment by eliminating the need for enzyme mediated labeling. Reproducibility of the data for high throughput drug screening is demonstrated by monitoring differential gene expression of a set of 45 gene targets involved in the genotoxic stress response pathways.