Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis

Abstract

DNA microarrays have the ability to analyze the expression of thousands of the same set of genes under at least two different experimental conditions1. However, DNA microarrays require substantial amounts of RNA to generate the probes, especially when bacterial RNA is used for hybridization (50 μg of bacterial total RNA contains approximately 2 μg of mRNA)2. We have developed a computer-based algorithm for prediction of the minimal number of primers to specifically anneal to all genes in a given genome. The algorithm predicts, for example, that 37 oligonucleotides should prime all genes in the Mycobacterium tuberculosis genome. We tested the usefulness of the genome-directed primers (GDPs) in comparison to random primers for gene expression profiling using DNA microarrays. Both types of primers were used to generate fluorescent-labeled probes and to hybridize to an array of 960 mycobacterial genes. Compared to random-primer probes, the GDP probes were more sensitive and more specific, especially when mammalian RNA samples were spiked with mycobacterial RNA. The GDPs were used for gene expression profiling of mycobacterial cultures grown to early log or stationary growth phases. This approach could be useful for accurate genome-wide expression analysis, especially for in vivo gene expression profiling, as well as directed amplification of sequenced genomes.

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Figure 1: (A) Flowchart depicting the algorithm used for developing genome-directed primers from bacterial genomes.
Figure 2: mtGDPs for measuring gene expressions.

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Acknowledgements

We wish to thank Walker Hale for excellent technical support, Chuck Epstein and Ross Chambers for helpful discussions. We particularly thank Susan Howard and Rick Lyons for providing RNA and DNA samples from M. tuberculosis H37Rv cultures. This work was supported by grants from DARPA and NIH to S.A.J. A.M.T. is supported in part by a cardiology training grant fellowship.

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Correspondence to Stephen Albert Johnston.

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Talaat, A., Hunter, P. & Johnston, S. Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis. Nat Biotechnol 18, 679–682 (2000). https://doi.org/10.1038/76543

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