DNA microarray

DNA microarray

A DNA microarray also known as DNA chip or a Biochip.

•            Definition - DNA microarrays are solid supports, usually of glass, nylon or silicon, on which Collection of microscopic DNA spots (DNA probes) are immobilized as microdots in an organized grid fashion. Each spot of DNA, called a probe, represents a single gene.

It allows the measurement of the level of gene expression for every gene in genome.

Principle

The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology, the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization.

Hybridization: The property of complementary nucleic acid sequences is to specifically pair

with each other by forming hydrogen bonds between complementary nucleotide base pairs.

Requirements - 

1. DNA chip
2. Target sample
3. Fluorescent dyes
4. Probes
5. Scanner
6. Enzymes

Steps -

1. Sample preparation
2. Purification (Isolate mRNA)
3. Reverse Transcription
4. Labeling
5. Hybridization
6. Scanning
7. Normalization and analysis.

Sample preparation - We’ll use two samples – cancerous human skin tissue & healthy human skin tissue.

Purification (Isolate mRNA) –

•         Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.
•         isolate the mRNA from total RNA by using affinity column containing beads with Poly-T tails to bind the mRNA. Rinse with buffer to release the mRNA from the beads.

Reverse Transcription & Labeling

•         Prepare cDNA by reverse transcription by using cyanine 3 (fluoresces green) labeled nucleotides for the healthy cell’s mRNA template and cyanine 5 (fluoresces red) labeled nucleotides for cancerous cell mRNA template. then finally degrade the mRNA.

Hybridization

•        Hybridize the labeled cDNA with DNA probes immobilized on microarray plate.
•        Computer data base having which gene contain in each spot.
•     after hybridizing wash to remove unhybridized cDNA.

Scanning

•         scanner consists with laser, a computer, and a camera.
•         The camera records the images produced when the laser scans the plate. The computer helps in view results & the store data. 

Normalization and analysis

• Green spots → cDNA from healthy tissue hybridized to the target DNA
• Red spots → cDNA from diseased/Cancer tissue hybridized to the target DNA (this is the interest result in disease/cancer diagnosis)
• Yellow spots → both healthy & diseased tissue cDNA hybridized equally to the target DNA (still the gene can carry out is function.
• Black spots → neither healthy or diseased tissue cDNA hybridized to the target DNA   

There are 2 types of DNA Chips/Microarrays - 

1. cDNA based microarray
2. Oligonucleotide based microarray

Applications-

1. Gene expression profiling –  In different cells/tissues, Under different environmental or chemical stimuli, In disease state versus healthy.
2. Discovery of drugs
3. Diagnostics – Microbial identification, microbial genotyping, Antibody detection, Cancer detection
4. Toxicological research (Toxicogenomics)
5. Pharmacogenomics - Individualized medicine.

For more information checkout this video -

•