DNA microarrays are solid supports usually made of glass or silicon upon which DNA is attached in an organized fashion. Each spot of DNA is called a probe represent a single gene. DNA Microarrays can analyze the expression of tens of thousands of genes simultaneously. DNA Microarray is also known as DNA chip.
The principle of DNA Microarray lies in the hybridization between nucleic acid strands and they pair with each other by forming Hydrogen bonds between them. Samples are labeled using a fluorescent dye and are hybridized to the chip. Complementary sequences between the sample and the probe get attached to the chip and paired via hydrogen bonds. Non-specific sequences that remain unattached can be washed during the process. Fluorescently labeled target sequences bind to the probe thereby generates a signal.
Two types of DNA Microarrays
- cDNA based Microarray
- Oligonucleotide based Microarray
cDNA based Microarrays
Chips are prepared using cDNA. cDNA is amplified using Polymerase Chain Reaction. They are immobilized on a solid support. Probe DNA loaded on a spotted spin by capillary action. A small volume of DNA preparation is spotted on a solid surface. DNA is delivered mechanically or in a robotic manner.
Small oligonucleotides spotted onto the array. Each gene is represented by more than one probe.
- DNA chip
- Target Sample
- Fluorescent Dyes
Healthy and Infected cell is culture and mRNA can be isolated using phenol-chloroform or using an organic solvent. mRNA is separated leaving behind rRNA and tRNA. mRNA has a poly-A tail. Column beads with poly T tails are used to bind mRNA. After extraction column is rinsed with buffer to isolate mRNA from beads. To create cDNA reverse transcription of mRNA is done. Both the samples are incorporated with different dyes to produce fluorescent DNA strands. Each cDNA gets hybridized to the complementary strand removing unwanted sequences. Data is collected using a microarray scanner. Scanner consists of laser, computer, camera. The laser excites the fluorescence of cDNA generating signals. The laser scans the array and the camera records the images produced. Computer stores the data and provides the data immediately. Data produced are analyzed and different intensities of colors for each spot determine the character of the gene.
Advantages of Microarrays
Microarray provides data of thousands of genes in real-time. This procedure is really fast and it is easy to obtain results. This technique is promising to discover diseases and cancer.