DNA Microarray
Study Notes
Definition
DNA microarrays are solid supports (usually glass or silicon) on which DNA is attached in an
organized, pre-determined grid fashion. Each spot, called a probe, represents a single gene.
They can analyze the expression of tens of thousands of genes simultaneously.
Also known as: DNA chips, gene chips, DNA arrays, gene arrays, biochips.
Principle
Based on hybridization between nucleic acid strands:
• Complementary nucleic acid sequences pair via hydrogen bonds between complementary
base pairs.
• Samples are labeled with fluorescent dyes.
• At least two samples are hybridized to the chip.
• Non-specific sequences remain unattached and are washed off.
• Fluorescently labeled sequences that bind to a probe generate a signal.
• Signal strength depends on hybridization conditions (e.g., temperature) and the amount of
target sample.
• Can screen 1 sequence in 100,000+ in a single hybridization.
Types of DNA Microarrays
1. cDNA-Based Microarray (Spotted DNA Arrays)
• Chips prepared using cDNA.
• cDNA is amplified by PCR.
• Immobilized on nylon filter or glass slide (1×3 inches).
• Probe DNA loaded into spotting pin by capillary action.
• Small volume spotted on solid surface by mechanical/robotic means.
2. Oligonucleotide Arrays (Gene Chips)
• Short DNA oligonucleotides (20-25 mers) spotted onto the array.
• Each gene is represented by more than one probe.
• Enabled by photolithography from the computer industry.
• Available off the shelf.
Requirements
• DNA Chip
• Target sample (fluorescently labelled)
• Fluorescent dyes
• Probes
• Scanner
Steps in cDNA-Based Microarray
Step 1: Collection of Samples
• Sample = cell or tissue of the organism under study.
• Two types collected: healthy cells and infected cells (for comparison).
Step 2: Isolation of mRNA
• RNA extracted using a column or solvent (e.g., phenol-chloroform).
• mRNA separated from rRNA and tRNA using poly-T-tail beads (binds poly-A tail of
mRNA).
• Column rinsed with buffer to isolate mRNA from beads.
Step 3: Creation of Labeled cDNA
• Reverse transcription of mRNA creates cDNA.
• Each sample labeled with a different fluorescent dye to distinguish them.
Step 4: Hybridization
• Labeled cDNAs placed on the microarray.
• Each cDNA hybridizes to its complementary strand.
• Array thoroughly washed to remove unbound sequences.
Step 5: Data Collection & Analysis
• Microarray scanner (laser + camera + computer) used to collect data.
• Laser excites fluorescence; camera records images; computer stores/analyzes data.
• Intensity difference in colors at each spot reveals gene expression character.
Applications
• Disease gene expression profiling in clinical medicine (diagnosis).
• Drug discovery.
• Diagnostics and genetic engineering.
• Alternative splicing detection.
• Proteomics.
• Functional genomics.
• DNA sequencing.
• Toxicological research (Toxicogenomics).
Advantages
• Provides data for thousands of genes simultaneously in real time.
• A single experiment generates many results.
• Fast and easy to obtain results.
• Promising for discovering cures for diseases and cancer.
• Different DNA parts can be used to study gene expression.
Disadvantages
• Expensive to create.
• Too many results at once — analysis is time-consuming and complex.
• DNA chips do not have a very long shelf life.