Table of Contents
- Introduction to DNA Microarray
- Principle of DNA Microarray Technique
- Types of DNA Microarrays
- Requirements of DNA Microarray Technique
- Steps Involved in cDNA based Microarray
- Applications of DNA Microarray
- Advantages of DNA Microarray
- Disadvantages of DNA Microarray
Introduction to DNA Microarray
- DNA microarrays are solid supports, usually made of glass or silicon, with DNA attached in an organized, pre-determined grid fashion.
- Each spot of DNA, called a probe, represents a single gene.
- DNA microarrays can analyze the expression of tens of thousands of genes simultaneously.
- Synonyms for DNA microarrays include DNA chips, gene chips, DNA arrays, gene arrays, and biochips.
Principle of DNA Microarray Technique
- The principle of DNA microarrays relies on the hybridization between nucleic acid strands.
- Complementary nucleic acid sequences specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.
- Samples are labeled using fluorescent dyes for the hybridization process.
- At least two samples are hybridized to the chip.
- Complementary nucleic acid sequences between the sample and the probe on the chip pair via hydrogen bonds.
- Non-specific bonding sequences remain unattached and are washed out during the washing step.
- Fluorescently labeled target sequences that bind to a probe sequence generate a signal.
- The signal depends on hybridization conditions (e.g., temperature) and washing after hybridization.
- The total strength of the signal depends on the amount of the target sample present.
- This technology can screen the presence of one genomic or cDNA sequence among 100,000 or more sequences in a single hybridization.
Types of DNA Microarrays
1. cDNA based microarray
2. Oligonucleotide based microarray
1. Spotted DNA arrays (“cDNA arrays”)
- Chips are prepared using cDNA.
- Called cDNA chips, cDNA microarray, or probe DNA.
- The cDNAs are amplified using PCR.
- These are immobilized on a solid support made of nylon filter or glass slide (1 x 3 inches).
- The probe DNA is loaded into a spotting pin by capillary action.
- A small volume of this DNA preparation is spotted on the solid surface, making physical contact between the two.
- DNA is delivered mechanically or in a robotic manner.
2. Oligonucleotide arrays (Gene Chips)
- In oligonucleotide microarrays, short DNA oligonucleotides are spotted onto the array.
- Small number of 20-25mers per gene.
- The main feature is that each gene is normally represented by more than one probe.
- Enabled by photolithography from the computer industry.
- Available off the shelf.
Requirements of DNA Microarray Technique
Requirements for Designing a DNA Microarray System:
- DNA Chip
- Target sample (Fluorescently labelled)
- Fluorescent dyes
- Probes
- Scanner
Steps Involved in cDNA based Microarray
The reaction procedure of DNA microarray takes places in several steps:
1. Collection of samples
- Collect samples from the organism, which may be cells or tissue.
- Obtain two types of samples: healthy cells and infected cells for comparison and results.
2. Isolation of mRNA
- Extract RNA from the sample using a column or solvent like phenol-chloroform.
- Separate mRNA from the extracted RNA, leaving behind rRNA and tRNA.
- Use column beads with poly-T-tails to bind the mRNA, which has a poly-A tail.
- Rinse the column with buffer to isolate mRNA from the beads.
3. Creation of labeled cDNA
- Perform reverse transcription of the mRNA to create cDNA (complementary DNA strand).
- Incorporate different fluorescent dyes into both samples to produce fluorescent cDNA strands, helping to distinguish the sample categories of the cDNAs.
4. Hybridization
- Place the labeled cDNAs from both samples in the DNA microarray to allow each cDNA to hybridize with its complementary strand.
- Thoroughly wash the microarray to remove unbounded sequences.
5. Collection and analysis
- Collect data using a microarray scanner, which consists of a laser, a computer, and a camera.
- The laser excites the fluorescence of the cDNA, generating signals.
- The camera records the images produced when the laser scans the array.
- The computer stores the data and provides results immediately.
- Analyze the data to determine the character of the gene at each spot based on the difference in the intensity of the colors.
Applications of DNA Microarray
Clinical Medicine:
- Determine how particular diseases affect the pattern of gene expression (expression profile) in various tissues.
- Identify the infecting organism from the expression profile.
- Significant potential for diagnosis.
Other Fields:
- Discovery of drugs
- Diagnostics and genetic engineering
- Alternative splicing detection
- Proteomics
- Functional genomics
- DNA sequencing
- Gene expression profiling
- Toxicological research (Toxicogenomics)
Advantages of DNA Microarray
- Provides real-time data for thousands of genes.
- A single experiment yields numerous results effortlessly.
- Quick and easy to obtain results.
- Promising tool for discovering cures for diseases and cancer.
- Enables the study of gene expression using different parts of DNA.
Disadvantages of DNA Microarray
- Expensive to create.
- Produces a large volume of results at once, requiring extensive and complex analysis.
- DNA chips have a limited shelf life.