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Chapter 18 : Solid- and Liquid-Phase Array Technologies
Category: Clinical Microbiology; Bacterial Pathogenesis
This chapter focuses on DNA-based hybridization array technology. The chapter reviews the methodologies of cDNA, oligonucleotide, electronic, and liquid bead arrays. In each of these methodologies, the probe refers to the DNA sequence bound to the solid surface support in the microarray, whereas the target is the ‘‘unknown’’ sequence of interest. Printed microarrays have the advantages of simplicity and relatively low cost compared to synthesized microarrays, which are discussed in the chapter. While two-dimensional microarrays have had an impact on our understanding of the microbial world and the application of this diagnostic potential to infectious diseases, the most widespread and practical application is the use of liquid bead suspension arrays in diagnostic microbiology, which is therefore emphasized when applicable. The potential use of low- or medium-density arrays for the simultaneous detection of large numbers of microbial genetic targets is one of the most promising areas in applying microarray technology to diagnostic microbiology. Broad-range amplification often focuses on the rRNA genes (16S, 18S, 23S, or intergenic transcribed spacers) due to the inherent dichotomy of conserved and polymorphic sequences. Though the range of organisms included was relatively limited (species of Bacillus, Listeria, Staphylococcus, Escherichia, Shigella, Klebsiella, Salmonella, Enterobacter, Ralstonia, Burkholderia, and Pseudomonas), this study provides proof of principle for the use of suspension arrays for the identification of broad-range amplification products. Though broad-range amplification typically focuses on the rRNA genes, other targets have been employed.
Key Concept Ranking
- Severe Acute Respiratory Syndrome
General workflow of printed microarrays. Probe preparation begins with the production of either denatured cDNA, genomic PCR products, or oligonucleotides that are subsequently spotted in an array format on a glass slide. In this example, two samples (one control and one experimental) are compared by extracting mRNA and converting the RNA sets into differentially labeled cDNA sets for hybridization. The inclusion of multiple fluorescent labels allows color differentiation based on the quantity of target cDNA from each set that hybridizes to the microarray probes. The fluorescence signal is then scanned and analyzed. Adapted and reprinted from reference 17 with permission from Elsevier.
GeneChip oligonucleotide microarray. (Top) Photolithography. UV light is passed through a lithographic mask that acts as a filter to either transmit or block the light from the chemically protected quartz wafer. Multiple lithographic masks are applied sequentially to determine the sequence synthesis on the microarray surface. (Middle) Chemical synthesis cycle. As the mask-filtered UV light removes the protecting groups (squares), a single nucleotide washed over the microarray surface is able to couple to the deprotected oligonucleotide chains. Sequential rounds of nucleotide addition combined with changes in the masks form a quartz wafer with 25-mers of predetermined sequence. (Bottom) Dicing and cartridge assembly. Once the wafer is completely synthesized, it can be diced into 49 to 400 individual microarrays, each then packaged into a plastic cartridge. Adapted and reprinted from reference 34 with permission from Elsevier and Affymetrix.
Electronic microarray. (A) An electronically activated pad creates a positive current, which enables the movement and concentration of negatively charged DNA probes to the activated locations. Once in the targeted location, the probes are secured by streptavidin-biotin bonds. (B) Once the first probe is bound to its targeted site, its activated pad can be turned off and a different pad can be activated. The repeated application of a positive current to test pad sites allows the probes to be arrayed. (C) Details of the RVA ASR. Once the probes have been bound, extracted and amplified nucleic acids from a patient sample are added and allowed to passively hybridize. After hybridization, secondary probes specific for the targets of interest with a nonspecific detector sequence are added along with fluorescent detector oligonucleotides. A positive hybridization event is measured following washing steps to remove unhybridized nucleic acids. The use of multiple fluorophores allows multiple probes to be used per site. P1, parainfluenza virus 1; P2, parainfluenza virus 2; P3, parainfluenza virus 3; FB, influenza virus B; FA, influenza virus A; IC, internal control; BKGD, background. Images courtesy of Nanogen.
Liquid bead suspension microarray. (A) Microspheres 5.6 μm in diameter are filled with various concentrations of an infrared dye and a red dye to create 100 spectrally distinct beads. (B) Microspheres can then be used in a variety of assays depending on the ligand bound to the bead surface. (Upper inset) Suspension bead direct hybridization. The target is amplified using a biotinylated primer and subsequently denatured and hybridized to microspheres tagged with target-specific sequence probes. A positive hybridization reaction at the microsphere surface is detected using streptavidin-R-phycoerythrin. (Lower inset) Solution-based chemistries for microsphere capture. ASPE: 1, denaturation of target DNA in the presence of specific capture sequence-tagged primers; 2, annealing of target DNA and primers; 3, primer extension and incorporation of biotinylated dNTP; 4, capture sequence-tagged ASPE products. OLA: 1, denaturation of target DNA in the presence of capture sequence-tagged allele specific probes; 2, annealing of target DNA and probes in a reaction containing a DNA ligase and biotinylated reporter probe; 3, oligonucleotide ligation; 4, capture sequence-tagged OLA products. SBCE: 1, denaturation of target DNA in the presence of a capture sequence-tagged primer (in separate reactions for each allele); 2, annealing of target DNA and primers; 3, single-base primer extension with incorporation of biotinylated ddNTP; 4, capture sequence-tagged SBCE products that can be multiplexed for detection. (C) After hybridization with the target of interest, the microsphere suspension is analyzed using a flow cytometer. A red laser (635 nm) excites the impregnated dyes of the microspheres to determine the spectral identity of the bead and therefore the probe being analyzed. A green (532-nm) laser excites the reporter fluorochrome to quantify the probe-target reaction on the microsphere surface. Insets reprinted from reference 43 with permission from Elsevier. Other images courtesy of Luminex.
Summary of differentiating characteristics of microarray platforms
Summary of key characteristics of the commercially available microarray products for respiratory viral diagnosis a