DNA chip-based microbiology

Clinical Microbiology Newsletter Vol. 23, No. 13

July 1.2001

DNA Chip-Based Microbiology James Versalovic, M.D., Ph.D. Assistant Professor ofPathology Harvard Medical School Director ofMolecular Diagnostics Massachusetts General Hospital Molecular Diagnostics Laboratory GRJ 529 55 Fruit St. Boston. MA 021/4

Introduction "DNA chips" refers to the immobilization of specific DNA probes on miniature solid surfaces. DNA microarrays are high density grids containing thousands of individual DNA probes and represent an important development in DNA chip technology (reviewed in reference 1). Microarrays enable largescale parallel hybridization experiments for global assessment of gene expression and sequencing (2). Macroarrays such as "dot blots" and "slot blots" preceded microarrays and permitted simultaneous analysis of multiple genetic targets on nylon or nitrocellulose membranes. In contrast to macroarrays, high-density DNA microarrays require microscopic analysis of fluorescent signals generated by nucleic acid hybridization of labeled target molecules and immobilized DNA probes. Following nucleic acid hybridization, fluorescent signals may be detected by confocal epifluorescence microscopy, permitting the increased resolution necessary for microarray image analysis. Either of two microarray formats is used by most laboratories and differ with respect to the probes, "spotted" cDNA arrays, or oligonucleotide arrays. Immobilized DNA probes (50 to 1,000 bases in length) consist of either ampIiClinicalMicrobiology Newsletter 23:13,2001

fied or cloned DNA that can be stored in microtiter plates. Microquantities of pre-synthesized DNA probes can be delivered or "spotted" to precise locations on glass or plastic surfaces by robotic systems (3,4). Such arrayers use different microprinting strategies, such as inkjet-like spraying, fountain penlike microdispensing, and pin-and-ring approaches. Thousands of DNA probes may be deposited onto membranes or glass slides by "spotting" techniques. Glass slides lack autofluorescence and are commonly used for cDNA arrays. Popular fluorescent dyes include Cy3 and Cy5 and are used in eDNA-based expression profiling by two-color fluorescence. Advantages of spotted eDNA microarrays include versatility, ease of customization, and accessibility of technology. Spotted bacterial genome macroarrays (Panorama, Sigma-Genosys, St. Louis, MO) are commercially available in addition to microbial genome primer collections for cDNA microarray development (e.g., Array-Ready Oligo Sets, Operon Technologies, Alameda, CAl. Oligonucleotide probes (typically 10 to SOmers) may be synthesized in situ on silicon-glass wafers using photolithogaphic techniques (GeneChip, Affymetrix, Santa Clara, CA) (5). Ultraviolet light penetrates a photolithographic mask to enable cyclic photodeprotection and incubation, resulting in polydeoxynucleotide synthesiS in situ. Advantages of oligonucleotide microarrays include improved sensitivity and reproducibility with controlled synthesis of fixed-length oligomer DNA probes in situ. Incorporated fluorescein 'C 200I ElsevierScienceInc.

In This Issue DNA Chip-Based Microbiology •• 99 As the clinical laboratory evolves toward increased automation and the genomics era provides abundant nucleic acid target sequences. DNA chips and microarrays will become important research and diagnostic tools during the next decade. Although DNA chip platforms are not currently usedfor routine clinical testing nor FDA approvedfor specific applications. the emerging importance of arrays in basic and clinical microbiology is apparent. Clearly, further development oflow-cost microarrays and bioelectronic chips will increase the accessibility ofDNA chip applications for the diagnostic laboratory.

Practical Aspects of the Consolidation of Clinical Microbiology Services (Experiences and Recommendations): Part 1 ••••• 103 In this two-part article. the author discusses his experiences in consolidating three clinical microbiology laboratories into one laboratory serving the three hospitals in which they were locatedformerly. Issues described in this part include reasons for consolidation; advantages and disadvantages ofconsolidation; planningfor the event; and major issues encountered. including personnel. standardization ofprocedures and tests. and instrument selection. Part 2 ofthis article will appear in the next issue ofClinical Microbiology Newsletter.

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or streptavidin-Iabeled phycoerythrin may be included for fluorescent signal detection in single-color GeneChip applications. Of interest to microbiologists, transcriptome (expressed sequence) chips for Escherichia coli and Saccharomyces cerevisiae are commercially available (Affymetrix).

Expression Profiling Microarrays enable the simultaneous analysis of global patterns of gene expression in microorganisms or host cells (2,6). Expression profiling by either cDNA or oligonucleotide microarray analysis may be useful for basic studies of genetic circuits, gene or target discovery, or molecular diagnostics. Various methods of studying gene expression predate the development of microarrays. Examples of conventional approaches include Northern blot analysis, cDNA library sequencing, and serial analysis of gene expression (7). In contrast to older methods, microarray analysis permits parallel expression analysis of expressed sequences in entire genomes or transcriptomes. Genome-wide expression studies enable investigators to identify changes in expression of multiple genes, and sets of genes in regulatory networks can be evaluated. Both cDNA and oligonucleotide microarrays have been applied to the study of transcriptomes in model microorganisms and microbial pathogens. Bacteria lack polyadenylated (polyA) messenger RNA (mRNA) species so oligo (dT)-based strategies cannot be used for mRNA isolation. Fifty micrograms) of bacterial total RNA contains approximately 2 ~g of mRNA. Although strategies to separate mRNA from excess amounts of ribosomal RNA have been developed, most applications in bacteria can be satisfactorily

performed with bulk cellular RNA. Approaches such as RNeasy (Qiagen, Valencia, CA) permit convenient isolation of total cellular RNA in bacteria by silica-based adsorption. RNA can be labeled by direct psoralen-biotin labeling (8) or fluorophore incorporation during cDNA synthesis (4). Fluorophorelinked dNTPs (e.g., Cy3- or Cy5-dUTP) are incorporated readily during reverse transcriptase-mediated cDNA synthesis with random hexamers. Alternatively, genome-directed primers can be designed instead of random oligonucleotides for target labeling (9). Sequence information has been used to design a finite set (n = 37) ofGDPs in order to increase the sensitivity and specificity of mycobacterial target RNA labeling (9). For oligonucleotide arrays, RNA may be harvested, digested with DNase I, and tagged with biotinylated nucleotides. Subsequent detection is mediated by streptavidin-phycoerythrin labeling and single-color fluorescence, the strategy commonly used in GeneChip applications. Alternatively, fluoresceinlabeled nucleotides may be incorporated into GeneChip targets during T3 or T7 RNA polymerase-mediated transcript synthesis. Direct psoralen-biotin labeling was performed with RNA harvested from Streptococcus pneumoniae (8). Labeled RNA was hybridized with a combination oligonucleotide array developed by Affymetrix, containing approximately 64,000 different probes representing 100 S. pneumoniae and 106 Haemophilus injluenzae genes (8). Pneumococcal transcript mapping by oligonucleotide microarray analysis confirmed the increased sensitivity of microarrays relative to conventional Northern blot analysis (8). GeneChip analysis was able to detect as few as two transcripts per cell and detected the presence of

RNAs missed by Northern blot analysis. Specific induction of competenceassociated genes (competence-induced or cin operon) was documented following the exposure of bacteria to competence stimulating peptide (CSP). Similarly, a direct comparison of radiolabeled "spot blot" experiments with cDNA microarray analysis of the E. coli K-12 genome yielded consistent data (10). Well-studied induction systems, lac-IPTG and heat shock, demonstrated the increased specificity and reproducibility of cDNA microarrays coupled with fluorescent signal detection (10). Glass slide-based cDNA microarrays were used to study gene expression of the E. coli trp repressor regulon (II) and diverse genes affected by atmospheric conditions in Bacillus subtilis (12). Temporal patterns of gene expression during bacterial cell cycle progression were assessed with glass slide-based cDNA microarrays containing 2,966 predicted open reading frames (ORFs) of Caulobacter crescentus (13). Induction of gene expression by stimulation of a two-component regulatory system was reported in S. pneumoniae (14). An oligonucleotide microarray representing nearly 2,000 ORFs of pneumococci verified induction of 16 genes in the blpC regulon by a synthetic oligopeptide. Microarray studies have added insights into mechanisms of antimicrobial action and identification of possible drug targets. Isoniazid (INH)-mediated alterations of gene expression in Mycobacterium tuberculosis yielded known targets in the type II fatty acid synthase (FAS) complex (15). INH was added to liquid cultures ofINH-resistant organisms, and expression was monitored with a glass slide-based cDNA array containing 3,834 (97%) of 3,924 ORFs. Genes not previously linked to INH

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resistance were induced and may represent novel antimicrobial targets. The antimycobacterial agent ethionamide also inhibits mycolate biosynthesis and yielded a similar gene expression profile when INH-susceptible M. tuberculosis was treated with ethionamide. Importantly, drugs with similar modes of action yielded similar microarray expression profiles. During drug research and development, the modes of action of novel compounds could be evaluated by microarray-based gene expression studies. Investigation of host responses to infection by microarray analysis represents an interesting application of DNA chips. Alterations of gene expression during infection may yield an increased understanding of microbial pathogenetic mechanisms. Diagnostic microbiology may be bolstered by the ability to simultaneously detect specific pathogens and associated host responses to infection (16). Common patterns of human gene expression during infection, which are assessed on a genome-wide scale, may become important facets in diagnostic laboratory medicine and provide useful information for disease diagnosis. Gene expression profiles induced by systemic infections caused by a number of different pathogens may converge to generate common host responses. When combined with pathogen detection, appropriate strategies for microbial elimination and down-modulation of destructive host responses may be determined by array-based diagnostics. Microarrays have been used to assess global responses to infection with human cytomegalovirus (17), coxsackievirus B3 (18), Ebola virus (19), human immunodeficiency virus type 1 (HIV-l) (20), Helicobacter pylori (21), Listeria monocytogenes (22), and Pseudomonas aeruginosa (23). Many other examples are being added to this list as microarray-based analysis of host gene expression in response to infection is studied with a variety of microbial pathogens.

Genotyping and Sequencing by Hybridization In addition to gene expression studies, microarrays may be used for organism identification and molecular resistance testing. Genotyping or sequencing by hybridization with oligonucleotide microarrays may proClinical Microbiology Newsletter23:13.200I

vide alternatives to capillary- or slab gel-based sequencing strategies (5,24). Microarrays with oligonucleotides containing 16S rDNA sequence information and antimicrobial target sequences facilitate simultaneous pathogen identification and molecular resistance testing. Oligonucleotide arrays interrogating 705 of 3,534 nucleotides of the M. tuberculosis rpoB gene distinguished 10 mycobacterial species and identified mutations in rifampin-resistant M tuberculosis isolates (25). Dideoxy DNA sequencing and array-based hybridization yielded complete concordance for identifiable mutations in rpoB associated with rifampin resistance. Importantly, mycobacterial species identification and clustering of 121 isolates by array-based hybridization of rpoB sequences were nearly identical to the groupings obtained by dideoxy sequencing. In addition to single-gene arrays, multiple-gene arrays have been developed. An oligonucleotide microarray containing 16S rDNA, katG. and rpoB sequences was used for simultaneous mycobacterial species identification and molecular resistance testing (26). This multiplex array, or array tiling strategy, illustrates the potential versatility of micro arrays as four different genetic targets were topologically separated within a 1.28 em' oligonucleotide microarray. The 20mer oligonucleotide probes interrogated a 169 nucleotide region in 16S rDNA, 51 mutations in a 200 bp region of rpoli, and a 2.2 kb region of katG. Array-based hybridization correctly identified 26 of 27 mycobacterial species and effectively distinguished closely related species. Future downward pricing shifts and advances in combination testing will increase the demand for multiplex DNA chip testing. The lIIV PRT GeneChip contains greater than 16,000 oligonucleotide probes for analysis of 40 codons in the lIIV-l protease and reverse transcriptase genes, associated previously with antiretroviral resistance (27). The HlV PRT GeneChip and dideoxy sequencing yielded excellent concordance (96.7%) with IIIV-l isolates from naive and treated patients (27). All primary mutations associated with treatment failure were identified by the IIIV PRT GeneChip. Discordant results were obtained with secondary mutations, but c,2ool ElsevierScience Inc.

both approaches were comparable overall for lIIV-l mutation detection in multiple studies (27,28). The emergence of non-clade B HlV-l isolates in Europe and North America poses a challenge for the mv PRT GeneChip as it is currently configured. Multiple base-calling ambiguities were reported when nonclade B lIIV-l isolates were examined with the lIIV PRT chip (29). With respect to molecular resistance testing, serine codon insertions distal to codon 68 in the RT gene were not detected by the mv PRT GeneChip (29).

Bioelectronic Chips The fusion of technologies in molecular biology and the semiconductor industry is contributing to the development of DNA chips and bioelectronics. Nanogen (San Diego, CAl is developing the NanoChip line, microelectronics chips that electronically couple DNA molecules to the chip surface and may facilitate easier customization of chips for diagnostic purposes. Infectious disease applications remain in development. Clinical Microsensors (Pasadena, CAl is developing portable, handheld devices to detect bioelectronic signals emitted by hybridization on relatively inexpensive DNA chips. This bioelectronic detection platform was used to detect the presence of the H63D polymorphism in the human hemachromatosis-associated HIe gene (30). Electrode arrays modified with specific DNA capture probes which target specific microbial pathogens may be developed. Hybridization of target nucleic acid with capture probes culminates in electronic signals detected by voltammetric analysis of electrode arrays. The combination of potentially mass-produced bioelectronic chips with lower unit production costs than current oligonucleotide-based microarrays and portable instrumentation may provide DNA chips attractive to clinical laboratories and point-of-care settings.

Summary and Future Directions As the clinical laboratory evolves toward increased automation and the genomics era provides abundant nucleic acid target sequences, DNA chips and microarrays will become important research and diagnostic tools during the next decade. Although DNA chip platforms are not currently used for routine clinical testing or str FDA approved for 0196-4399/00(see frontmatter)

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specific applications, the emerging importance of arrays in basic and clinical microbiology is apparent. Genomewide analyses with microarrays are yielding tremendous amounts of information in research efforts exploring microbial pathogenesis and associated host responses. Microarrays may indicate shared mechanisms of action among antimicrobial agents and facilitate drug discovery efforts in industry. Simultaneous pathogen identification and resistance testing using combination arrays may increase the role of the clinical laboratory in timely clinical management. An enhanced ability to detect microbial pathogens and globally assess human responses to infection may increase the role of the diagnostic laboratory in patient management. Finally, further development of lowcost microarrays and bioelectronic chips will increase the accessibility of DNA chip applications for the diagnostic laboratory. References

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