MEVE 018: Unit 13 – Microarrays

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UNIT 13: MICROARRAYS

13.0 Introduction

The rapid advancement of molecular biology and genomics has enabled researchers to study gene expression on a genome-wide scale. Microarray technology is one such powerful tool that allows simultaneous analysis of thousands of genes, offering insights into gene function, interaction, and expression patterns under various environmental conditions. This technique has become increasingly valuable in environmental biotechnology for monitoring pollutants, studying microbial communities, and understanding responses to environmental stress.

13.1 Objectives

After studying this unit, you should be able to:

  • Understand the history and development of DNA microarrays.
  • Explain the steps in preparing DNA microarrays.
  • Identify different types of microarrays.
  • Compare short and long oligonucleotide arrays.
  • Discuss the advantages and disadvantages of oligonucleotide arrays.
  • Explore the applications of microarrays in environmental studies.

13.2 History of DNA Microarray

The origins of microarray technology date back to the 1990s when researchers sought high-throughput techniques to monitor gene expression. Initially developed by the Stanford University group (Patrick Brown and colleagues), the first microarrays used glass slides with immobilized DNA probes. Over time, the technology evolved to include oligonucleotide arrays and commercial platforms like Affymetrix and Agilent, which allowed better accuracy and wider applications.

13.3 Substrates Used for Microarray Fabrication

Microarrays are fabricated on solid supports, which provide a platform for immobilizing DNA probes. Common substrates include:

  • Glass slides – the most commonly used due to their optical clarity and chemical stability.
  • Nylon membranes – used for radioactive labeling and hybridization.
  • Silicon chips – used in high-density commercial arrays.

The substrate is chemically treated to enhance binding between the DNA probes and the surface.

13.4 Preparation of DNA Arrays

13.4.1 Sample Preparation and Labelling

  1. Total RNA or mRNA is extracted from the sample.
  2. Reverse transcription is performed to synthesize complementary DNA (cDNA).
  3. The cDNA is labeled with fluorescent dyes (e.g., Cy3, Cy5) for detection.
  4. The labeled DNA is purified for hybridization.

13.4.2 Array Hybridisation

The labeled cDNA sample is applied to the microarray slide where it hybridizes with complementary DNA probes fixed on the surface. This hybridization occurs under specific temperature and buffer conditions.

13.4.3 Image Acquisition

Post-hybridization, the slide is washed to remove non-specifically bound DNA. It is then scanned using a laser scanner that detects fluorescent signals, which reflect the amount of hybridization at each spot. The data is processed using specialized software for analysis.

13.5 Types of DNA Microarrays

13.5.1 Glass cDNA Microarrays

  • Contain PCR-amplified cDNA fragments spotted on glass slides.
  • Used to analyze gene expression profiles.
  • Relatively inexpensive and customizable.

13.5.2 Oligonucleotide Microarrays / In Situ Oligonucleotide Arrays

  • Use short synthetic DNA sequences (oligonucleotides) synthesized directly on the chip surface.
  • High specificity due to uniform probe length.
  • Suitable for SNP detection, gene expression, and genotyping.

13.6 Advantages of Microarray

  • Simultaneous analysis of thousands of genes.
  • High sensitivity and specificity.
  • Useful in toxicogenomics and ecological studies.
  • Can detect gene expression changes in response to pollutants or stress.
  • Helps in functional genomics and systems biology.

13.7 Oligonucleotide Arrays

Oligonucleotide arrays use synthetic short DNA fragments as probes. These can be designed to target specific genes or mutations.

13.7.1 Short Oligonucleotide Arrays: In Situ Synthesis

  • Typically 25–60 nucleotides long.
  • Synthesized base by base directly on the chip surface using photolithographic or inkjet printing methods.
  • Example: Affymetrix GeneChip.

13.7.2 Long Oligonucleotide Arrays: In Situ Synthesis

  • Typically 60–100 nucleotides.
  • Offer higher binding strength and sensitivity.
  • Suitable for detecting low-abundance transcripts.
  • Used by companies like Agilent Technologies.

13.8 Advantages of Oligonucleotide Arrays

  • High specificity due to shorter, unique sequences.
  • Greater reproducibility and consistency.
  • Easier to design and customize.
  • Lower background noise in hybridization.
  • Ideal for comparative genomic hybridization (CGH) and mutation detection.

13.9 Disadvantages of Oligonucleotide Arrays

  • Short probes may result in weaker hybridization signals.
  • Require high-quality sequence information.
  • Costlier than traditional cDNA arrays.
  • Lower sensitivity compared to longer DNA probes for low-expressing genes.

13.10 Applications of Microarrays in Environmental Studies

Microarrays have become essential tools in environmental genomics and toxicology.

Key Applications:

  1. Microbial Community Analysis:
    • Study biodiversity and population dynamics in soil, water, and waste systems.
  2. Pollution Monitoring:
    • Detect changes in gene expression in organisms exposed to pollutants.
    • Identify biomarkers for heavy metal or pesticide exposure.
  3. Biodegradation Studies:
    • Monitor genes involved in the breakdown of organic pollutants.
  4. Ecotoxicology:
    • Assess the genetic impact of toxins on aquatic and terrestrial organisms.
  5. Bioremediation:
    • Identify and track genes in microbes involved in pollutant degradation.

13.11 Applications of Microarrays (General)

  • Medical diagnostics (e.g., cancer profiling, infectious disease detection).
  • Pharmacogenomics (drug response prediction).
  • Agriculture (GMO screening, stress gene analysis).
  • Functional genomics (gene discovery and regulation studies).

13.12 Let Us Sum Up

DNA microarrays are advanced molecular tools for studying gene expression on a large scale. This unit covered the evolution of microarrays, fabrication techniques, and types of DNA arrays, especially oligonucleotide arrays. Their environmental applications make them valuable in understanding how organisms respond to pollution and stress, and how ecosystems adapt or degrade under anthropogenic influences.

13.13 Glossary

  • Microarray-A grid of DNA probes used to measure gene expression or genetic variation.
  • Hybridization-Binding of complementary DNA strands on a microarray.
  • cDNA-Complementary DNA synthesized from mRNA using reverse transcriptase.
  • Fluorescent labeling-Tagging of DNA with fluorescent dyes to detect hybridization.
  • Oligonucleotide-A short DNA or RNA molecule, typically 20–100 bases long.
  • Affymetrix-A company known for manufacturing commercial oligonucleotide arrays.
  • Gene expression-The process by which information from a gene is used to synthesize a functional product.
  • Toxicogenomics-Study of the effects of toxic chemicals on gene expression.

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