DNA microarrays contain thousands of DNA probes attached to a solid surface that are used to simultaneously measure gene expression levels in a sample. The sample's DNA or RNA is extracted, labeled, and hybridized to the microarray to bind to complementary probes. A scanner then detects the bound material's fluorescence intensity to quantify gene expression levels. This allows researchers to analyze expression patterns and identify genes that are differently expressed under different conditions, such as disease states or drug treatments.

1. TITLE: DNA MICROARRAY
SUBMITTED BY: HAJRA BIBI
ROLL NUMBER: 23011760-018
SUBMITTED TO: Dr. MUDDASSIR ZAFAR
COURSE TITLE: ADVANCE RESEARCH
TECHNIQUES
COURSE CODE: BCH-512
SECTION: MPHIL BIOCHEM-A
DEPARTMENT OF BIOCHEMISTRY
UNIVERSITY OF GUJRAT HAFIZ HAYAT CAMPUS

2. INTRODUCTION
A DNA microarray, also known as a DNA chip, gene chip, or biochip, is a small glass slide or
silicon chip that contains thousands of microscopic spots of DNA attached to its surface. Each
spot, which is only a few microns in diameter, contains a specific DNA sequence, which can be a
gene, part of a gene, or another regulatory element.
DNA microarrays are used to measure the expression levels of thousands of genes
simultaneously. This is done by hybridizing a sample of labeled DNA or RNA to the microarray.
The labeled DNA or RNA will bind to the complementary DNA sequences on the microarray,
and the amount of bound DNA or RNA can be measured using a variety of methods, such as
fluorescence or radioactivity.
PRINCIPLE
The Principle of DNA microarray technology is the capacity of complementary DNA strands to
hybridize, or bond, with one another. A biological sample’s complementary DNA sequences are
intended to hybridize with the little pieces of DNA, or probes that make up a DNA microarray.
The joining of two complementary DNA strands by hydrogen bonding between the nucleotide
base pairs is the process of DNA hybridization. DNA is composed of the nucleotide bases
adenine (A), thymine (T), cytosine (C), and guanine (G). Adenine always pairs with thymine,
and cytosine always pairs with guanine, to form base pairs.
INSTRUMENTATION
Several types of equipment are needed for the DNA microarray approach, and they are employed
at various phases of the process. Here are some of the major instruments used in DNA
microarray technique:
1. Microarray printer: DNA probes are created and printed onto a glass slide or another
solid surface using a microarray printer. A pattern of probes that can be used to measure gene
expression levels may be created by programming the printer to drop small droplets of DNA
solution onto the slide.
2. Hybridization oven: The microarray slide and the labeled sample are incubated together
in a hybridization oven. The hybridization of the probes and sample is made possible by the
oven’s regulated temperature and humidity conditions.
3. Microarray scanner: The fluorescent signals produced by the hybridized probes and
labeled material are picked up by a microarray scanner. A quantitative measurement of the
levels of gene expression may be obtained from the scanner by detecting the fluorescence
intensity that each probe on the slide emits.
4. Data analysis software: The microarray scanner’s data is analyzed using specialized
software. The program can provide graphical representations of gene expression patterns,
normalize the data, and remove noise.
5. Real-time PCR machine: The outcomes of a DNA microarray study can be verified
using a real-time PCR equipment. Real-time PCR is able to measure the sample’s expression
levels of certain genes and validate the outcomes of microarray analysis.

3. MECHANISM
DNA microarrays, those microscopic marvels of genetic analysis, operate through a series of
meticulously orchestrated molecular events. Here's a deeper dive into the mechanism, using
scientific terms for greater precision:
1. Preparation of the Stage:
 Solid Substrate: The foundation of a microarray is a solid substrate, typically glass or
silicon. This chip is chemically treated to facilitate the covalent attachment of DNA
probes.
 Probe Immobilization: Oligonucleotides, short DNA sequences complementary to
target genes or regulatory elements, are covalently linked to the chip surface. These
probes, often synthesized via photolithography or inkjet printing, represent thousands of
unique DNA sequences, creating a miniaturized library on the chip.
2. Target Hybridization:
 Sample Extraction and Labeling: RNA or DNA is extracted from the biological sample
of interest. This target nucleic acid is then labeled with a fluorescent dye or other reporter
molecule, enabling its detection after hybridization. Common labeling methods include
fluorescent dyes (Cy3/Cy5), biotin-streptavidin conjugation, and enzymatic
amplification.
 Hybridization: The labeled target is applied to the microarray chip and incubated under
controlled conditions. During this phase, the target molecules undergo
hybridization, seeking and binding to their complementary probes on the chip via
Watson-Crick base pairing. The specificity of this interaction ensures accurate target
identification.
3. Signal Amplification and Detection:
 Washing and Stringency: Unbound target molecules are carefully washed away, leaving
only the specifically hybridized pairs on the chip. The washing stringency can be adjusted
to control the level of hybridization specificity, optimizing for sensitivity or accuracy
depending on the experimental goals.
 Signal Detection: Depending on the chosen reporter molecule, different techniques are
employed for signal detection and quantification. Fluorescence scanners capture the
emission of fluorescent dyes at specific wavelengths, while chemiluminescent or
enzymatic reporters may require additional processing steps.
4. Data Analysis and Interpretation:
 Image Acquisition and Processing: Scanned microarray images are converted into
digital data, with each spot representing the intensity of the hybridization signal at that
location. Data processing algorithms normalize and analyze the fluorescence
intensities, accounting for background noise and technical variations.
 Statistical Analysis and Gene Expression Profiling: Statistical tests are performed to
identify differentially expressed genes between samples or experimental conditions. This
analysis reveals alterations in gene activity patterns, providing insights into cellular
processes, disease mechanisms, or treatment responses.

4. INTERPRETATION OF RESULTS
1. Data Acquisition and Preprocessing:
 Image Scanning and Processing: The microarray image is converted into digital
data, with each spot representing the intensity of the hybridization signal (usually
fluorescence).
 Quality Control: Check for artifacts, background noise, and technical variations to
ensure data integrity.
 Normalization: Adjust raw intensity values to account for systematic differences
between arrays or samples, allowing for meaningful comparisons.
2. Data Analysis and Visualization:
 Statistical Analysis: Employ statistical methods to identify differentially expressed
genes between experimental conditions or samples.
 Fold Change: Calculate the magnitude of gene expression changes, indicating how much
a gene's expression level differs between groups.
 P-value: Assess the statistical significance of observed differences, determining the
likelihood they occurred by chance.
 Visualization: Create heatmaps, scatter plots, or hierarchical clustering diagrams to
visualize gene expression patterns and relationships.
3. Biological Interpretation:
 Functional Annotation: Link differentially expressed genes to their known biological
functions, pathways, and networks.
 Literature Search: Explore existing research to understand the roles of identified genes
in relevant biological processes or diseases.
 Pathway Analysis: Examine how differentially expressed genes interact within cellular
pathways to gain insights into broader biological mechanisms.
 Validation: Confirm microarray results using independent methods like quantitative
PCR (qPCR) or RNA sequencing for enhanced confidence.
4. Application-Specific Considerations:
 Gene Expression Profiling: Focus on identifying genes with altered expression patterns
associated with specific conditions or treatments.
 Genotyping: Identify individual genetic variations (SNPs, mutations) related to disease
susceptibility or drug response.
 Comparative Genomic Hybridization (CGH): Detect chromosomal copy number
changes indicative of tumor development or genetic disorders.
5. Integration with Other Data:
 Combine microarray data with other omics data (proteomics, metabolomics) for a more
comprehensive understanding of biological systems.
 Incorporate clinical or phenotypic data to correlate gene expression patterns with patient
outcomes or disease characteristics.
FLOW SHEET

5. APPLICATIONS
DNA microarray technology has a wide range of applications, including:
1. Gene expression profiling: This is the most common use of DNA microarrays. It
allows researchers to measure the expression levels of thousands of genes in a single
experiment. This can be used to study how gene expression changes in response to
different conditions, such as disease, development, or treatment with a drug. DNA
microarrays are often used to compare the levels of gene expression in various samples,
such as healthy and sick tissues, in order to pinpoint the genes that are up- or down-
regulated under particular circumstances.
2. Genotyping: DNA microarrays can also be used to genotype DNA samples. This
means that they can be used to identify the specific alleles of a gene that are present in an
individual. This can be used for a variety of purposes, such as diagnosing genetic diseases
or identifying individuals who are at risk of developing a particular disease.
3. Comparative genomic hybridization (CGH): CGH is a technique that can be
used to identify chromosomal imbalances in cancer cells. This is done by hybridizing
labeled DNA from a tumor sample and a normal DNA sample to a microarray. The
amount of bound DNA from each sample will be different if there are any chromosomal
imbalances in the tumor cells.
4. Drug discovery: DNA microarrays can be utilized to identify compounds that target
certain genes or pathways and to evaluate possible therapeutic candidates for their
influence on gene expression. With the use of DNA microarrays, clinicians may now
customize medicines for specific patients in light of their genetic profiles, opening up
new possibilities for customized medicine.
5. Microbial analysis: Based on their genetic profiles, DNA microarrays may be used
to identify and categorize microorganisms. DNA microarrays have been used to track the
diversity and activity of microbes in a variety of environmental samples, including soil
and water, revealing information on how human activities affect the environment.
6. Forensic analysis: DNA microarrays can be used in paternity tests or criminal
investigations to identify people based on their genetic characteristics.
7. Agricultural research: DNA microarrays have been used to increase agricultural
yields and create new strains of crops that include desired features like insect resistance
or a tolerance for environmental challenges.