DNA microarray

DNA microarray is a powerful high-throughput technology that enables the

simultaneous analysis of thousands of genes. It provides a comprehensive view of both
the genome and the transcriptome, allowing scientists to better understand the
molecular basis of normal and abnormal biological processes.

This technology significantly accelerates the screening and analysis of large numbers
of DNA or RNA samples and is widely used in gene expression studies, disease
research, and genomics.

Basic concept of DNA microarray:

• A typical microarray experiment involves the hybridization of an mRNA molecule

to its DNA template.
• Many DNA samples are used to construct the array.
• The amount of mRNA bound to each site on the array indicates the expression
level of the genes.
• Thousands of genes can be analysed at the same time.
• All the data is collected, and a profile is generated for gene expression of the cell.
• DNA microarray (also commonly known as gene chip, DNA chip, or biochip)
• A DNA microarray is a collection of microscopic DNA spots attached to a solid
surface.
• In DNA chip technology, single stranded DNA molecule is immobilized on solid
surface by biochemical analysis.
• These DNA molecules are derived from:
o cDNA sequences of an organism referred as cDNA microarray or
o Short oligonucleotides synthesized using photolithography
• Scientists use DNA microarrays to measure the:
expression levels of large numbers of genes simultaneously or to genotype
multiple regions of a genome.
• Each DNA spot contains picomoles (10"12 moles) of a specific DNA sequence,
known as probes (or reporters).
• These can be a short section of a gene or other DNA element that are used
to hybridize a cDNA or cRNA sample (called target) under high-stringency
conditions.
• Probe-target hybridization is usually detected and quantified by detection of
fluorophore , silver-, or chemiluminescence-labelled targets to determine
relative abundance of nucleic acid sequences in the target.
Principle of DNA microarray:

The fundamental principle of DNA microarray is based on nucleic acid hybridization,

which depends on the ability of complementary DNA strands to bind to each other
through hydrogen bonding between base pairs (A–T and G–C).

• The greater the complementarity between probe and target, the stronger the
hybridization.
• Only strongly matched sequences remain bound after washing steps.
• Hybridization strength is influenced by:
o Number of complementary bases
o Temperature
o Stringency conditions (washing, salt concentration)

The detected signal is directly proportional to the amount of target bound to the probe.

Experimental Workflow: Components of DNA Microarray:

[Link]
Step 1: RNA Extraction: mRNA is • Short or long single-stranded DNA
isolated from biological samples (e.g., sequences
healthy vs diseased cells) • Immobilized on a solid surface
Step 2: cDNA Synthesis and Labelling • Represent known genes or genomic
• mRNA is converted into cDNA
regions
• cDNA is labelled using
[Link]
fluorescent dyes or other markers • Labelled cDNA or cRNA derived from
Step 3: Hybridization mRNA
• The labelled target is applied to
• Represent the sample being analysed
the microarray [Link] Support
• Complementary sequences bind
• Glass slide or silicon chip
to probes • Provides a platform for probe
Step 4: Washing attachment
• Removes non-specific or weak
bindings Signal Interpretation and
• Ensures high specificity
Step 5: Detection Quantification
• Signals are detected using
• Signal intensity is proportional to
Fluorescence scanners,
target concentration
Chemiluminescence,
• Microarrays use relative
Autoradiography
quantification, meaning: Expression
Step 6: Data Analysis
levels are compared between
• Signal intensity at each spot is
different conditions
measured
• Each spot is identified by its fixed
• Reflects gene expression levels
position on the chip
Types of DNA Microarrays

1. Oligonucleotide-Based Microarrays
• Contain short DNA sequences (10–25 nucleotides)
• Synthesized directly on the chip using photolithography
• High-density arrays (up to hundreds of thousands of probes)
• Developed by Affymetrix
Advantages: High specificity, Precise probe design
Limitations: Expensive, technically complex

2. cDNA-Based Microarrays
• Use longer DNA fragments derived from mRNA
• Prepared by synthesizing cDNA and spotting it onto the chip
• Developed by Davis and Brown
Advantages: Easier and more cost-effective
Limitations: Lower specificity compared to oligonucleotide arrays, Requires cDNA or
EST libraries

3. Advanced Microarray Formats: Double-Stranded DNA Chips

• DNA probes are converted into double-stranded form
• Prepared using:
o Primer extension (Klenow fragment)
o Ligation on gold surfaces
• Allows interaction with proteins and accessibility
• Alternative methods include coupling oligonucleotides to gold surfaces and
ligating double-stranded DNA.

Hybridization and detection techniques:

Hybridized targets can be detected using: Fluorescent labelling (most common),
Radioactive labelling, Enzyme-based detection
Detection systems include Laser scanners, CCD-based imaging systems

Applications of DNA Microarray

1. Gene Expression Profiling
• Measures expression levels of thousands of genes
• Used to study: Disease conditions, Drug responses, Developmental stages
2. Toxicogenomic
• Studies the effects of toxic substances on gene expression
• Helps understand genetic responses to environmental stress
3. Comparative Genomic Hybridization
• Compares genomic differences between samples
• Identifies variations in genome content
4. Gene Identification (GeneID)
• Detects: Pathogens, GMOs, Contaminants (e.g., mycoplasma)
 • Often combined with PCR techniques
5. Chromatin Immunoprecipitation on Chip (ChIP-on-Chip)
• Identifies DNA regions bound by proteins
• Used in: Epigenetics, Transcription studies
6. DamID
• Alternative to ChIP
• Uses DNA methylation instead of antibodies
• Identifies protein-DNA interactions
7. SNP Detection
• Identifies single nucleotide polymorphisms
• Applications include Disease susceptibility, Cancer mutation analysis, Forensic
studies
8. Alternative Splicing Analysis
• Detects different splice variants of genes
• Uses exon-specific probes
9. Fusion Gene Detection
• Identifies gene fusions (important in cancer diagnosis)
10. Tiling Arrays
• High-resolution genome mapping
• Detects: Insertions, Deletions, Mutations
• Used in reverse genetics
11. Plant and Environmental Studies
• Analyses gene expression under different environmental conditions