DNA MICROARRAY- DEMONSTRATION
INRODUCTION:
In a DNA microarray experiment, thousands of DNA probes are placed on a solid surface to
assess the relative abundance of thousands of distinct mRNA molecules, which indicates the
levels of gene expression. Following cell collection, the mRNA from the cells is transformed
into complementary DNA (cDNA) that has been fluorescently tagged and placed to the array.
When complementary cDNA hybridizes with its corresponding probe, a fluorescence signal is
produced that can be used to identify the active genes.
DNA microarray is a high-throughput molecular technique used to simultaneously
analyze the expression of thousands of genes or detect specific DNA sequences.
It allows identification of gene expression profiles, genetic variations, or mutations in
a single experiment.
Microarrays are essential for functional genomics, disease diagnostics, drug
discovery, and personalized medicine.
The technique uses complementary base pairing to detect target DNA or RNA by
hybridization to immobilized probes on a solid surface.
Fluorescent or other labeled targets provide quantitative and qualitative information
about gene expression or sequence presence.
DNA microarrays help researchers compare normal vs. diseased cells, monitor
treatment effects, or study genome-wide regulation.
Microarray technology has been extensively used by the scientific community for the novel
applications. The main advantage of this method is the genomic wide information provided at
reasonable costs.
PRINCIPLE:
The core principle behind microarrays is hybridization between two DNA strands, the
property of complementary nucleic acid sequences to specifically pair with each other by
forming hydrogen bonds between complementary nucleotide base pairs. A high number of
complementary base pairs in a nucleotide sequence means tighter non-covalent bonding
between the two strands. After washing off of non-specific bonding sequences, only strongly
paired strands will remain hybridized. So fluorescently labelled target sequences that bind to
a probe sequence generate a signal that depends on the strength of the hybridization
determined by the number of paired bases, the hybridization conditions (such as temperature),
and washing after hybridization. Total strength of the signal, from a spot (feature), depends
upon the amount of target sample binding to the probes present on that spot. Here the
unknown sample of DNA sequence is referred as the target or sample and known sequence of
DNA molecule which is referred as probe. After completion of hybridization the surface of
chip can be analyze quantitatively and qualitatively by using autoradiography, laser scanning,
fluorescence detection device, enzyme detection system.
Applications:
1. Studying global gene expression patterns in normal vs. diseased tissues.
2. Identification of biomarkers for cancer and other diseases.
3. Screening for mutations, SNPs, and genetic variations.
4. Evaluating drug responses at the molecular level.
5. Functional genomics studies to understand gene regulation and networks.
6. Comparative genomics in different species or cell types.
Fig: Steps involved in DNA Microarray
MATERIALS / REAGENTS:
DNA microarray chip with immobilized probes
RNA or cDNA sample from the cells/tissue
Reverse transcriptase and labeling dyes (fluorescent dyes like Cy3/Cy5)
Hybridization buffer
Microarray hybridization chamber
Washing buffers
Microarray scanner and software for data analysis
Gloves, micropipettes, and sterile tips
PROTOCOL:
Step 1: Sample Preparation and Labelling
1. Isolate total RNA from cells or tissue.
2. Convert RNA to cDNA using reverse transcriptase.
3. Label cDNA with fluorescent dyes (e.g., Cy3 for control, Cy5 for test sample).
Step 2: Hybridization
1. Apply labelled cDNA to the microarray chip.
2. Place the chip in a hybridization chamber to prevent evaporation.
3. Incubate at 42–65°C for several hours to allow specific probe-target binding.
Step 3: Washing
1. Wash the chip carefully with stringent washing buffers to remove non-specifically
bound targets.
Step 4: Detection
1. Scan the microarray chip using a microarray scanner.
2. Fluorescent spots indicate hybridization; intensity correlates with gene expression
level.
Step 5: Data Analysis
Use software to quantify fluorescence intensity and generate gene expression profiles.
Compare test and control samples to identify upregulated or downregulated genes.
INTERPRETATIONS:
Bright fluorescent spots indicate genes that are highly expressed or present in the
sample.
Dim or absent spots indicate low or no expression.
Comparison between samples allows identification of differentially expressed genes.
RESULT:
CONCLUSION:
