Comparative and Functional Genomics

Project Based Learning

2023

Submitted to – Prof. Vibha Rani

Submitted by –
Divyansh Upadhyay
C2
20101047

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 MICRO ARRAY

A microarray is a tool used in molecular biology and genetics to assess the simultaneous
expression levels of many genes. It consists of thousands of small DNA or RNA molecules
bonded to tiny silicon chips or glass slides in an organised grid. These small substances,
sometimes referred to as probes, complement certain DNA sequences that are present in the
genome for designated genes.
For a microarray experiment, researchers use RNA from a sample of cells or tissue and reverse-
transcribe it to create complementary DNA (cDNA). The cDNA can bind to the complementary
probes on the chip after being hybridised to the microarray and tagged with a fluorescent dye.
The fluorescence signal intensity at each probe position is analysed to quantify the level of
related gene expression in the sample.
Microarrays can be used to investigate a wide range of biological processes, including gene
expression, gene regulation, protein-protein interactions, and epigenetic changes. They are a
useful technique for identifying genes whose expression differs between samples, such as
healthy tissue and sick tissue or normal conditions and experimental settings. Our
understanding of many diseases, including cancer, diabetes, and heart disease, has increased
because to the intensive use of microarrays in genomics research.
Numerous biological processes, such as gene expression, gene regulation, protein-protein
interactions, and epigenetic modifications, can be studied using microarrays. They are a
practical method for locating genes whose expression varies between samples, such as between
healthy tissue and sick tissue or between normal conditions and experimental settings. The
extensive use of microarrays in genomics research has improved our understanding of
numerous diseases, such as cancer, diabetes, and heart disease.

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TECHNIQUE –
• • To conduct a microarray analysis, mRNA molecules are normally extracted from
both an experimental sample and a reference sample. For instance, the reference
sample might be drawn from a healthy person, while the experimental sample might
be drawn from a cancer patient. A fluorescent probe of a different hue is subsequently
used to mark the complementary DNA (cDNA), which is produced as a result of the
conversion of the two mRNA samples.

Contrarily, the experimental cDNA sample may be labelled with a red fluorescent dye
whereas the reference cDNA may be marked with a green fluorescent dye. The two
samples are then blended, and time is allowed for them to attach to the microarray
plate.

• The way that the cDNA molecules bind to the DNA probes on the slide is by
hybridization. After hybridization, the microarray is scanned to ascertain the level of
expression for each gene shown on the slide. If the expression of a particular gene is
higher in the experimental sample than in the reference sample, the relevant location
on the microarray turns red. However, if the expression in the experimental sample is
lower than in the reference sample, the spot appears green.

Finally, the spot will look yellow if the two samples have equal expression. The data
gathered by microarrays can be used to create gene expression profiles, which show
simultaneous changes in the expression of many genes in response to a certain
stimulus.

• The ability to deposit tens of thousands of DNA sequences on a tiny surface,

frequently a glass slide (sometimes referred to as a "chip"), forms the basis of gene
microarray technology. So that each DNA fragment's identification may be
ascertained by its position on the array, the numerous DNA fragments are arranged in
rows and columns.

• Tissue microarrays (TMA) and gene expression microarrays are two different types of
microarrays. Using techniques like Northern blot and reverse transcriptase-
polymerase chain reaction (RT-PCR), just a few genes can be examined in a single
experiment. The advantage of microarray or "global expression profiling" is that the
genes under evaluation are not impacted by gene preselection. Additionally, it
evaluates orders of magnitude more genes than it did in the past.

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PRINCIPLE –
The intermediary molecule RNA transports the genetic material required to produce proteins
from the cell nucleus to the cytoplasm. When a gene is expressed or in its active state,
transcription takes place and many copies of the mRNA corresponding to that gene are
produced.
The appropriate protein is made by these mRNAs. As a result, by assessing the various
mRNAs, we can indirectly assess the genetic data or the expression of genes. This makes it
easier to understand the various mechanisms that underlie each changed genomic expression.
Consequently, mRNA acts as a substitute marker. It is necessary to convert mRNA into the
more lasting cDNA form since mRNA is easily degraded. cDNA is marked with the
fluorochrome dyes Cy3 (green) and Cy5 (red).
Restrictions endonucleases fragment the unidentified DNA molecules, which are then
labelled with fluorescent dyes. These are then given a chance to interact with DNA chip
probes. When complimentary sequences are present, the target DNA fragments attach to the
DNA probes. The last bits of DNA are washed away. When a laser beam passes over the
target DNA fragments, its fluorescence emission can be used to identify them. The pattern of
fluorescence emission and DNA identification are recorded using a computer. This method of
using DNA chips is particularly quick and sensitive and specific for concurrently identifying
several DNA fragments.

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The majority of investigations on the expression of a specimen's genes are no longer
hypothesis-driven, but rather are referred to as "discovery-type research" or, in a less
complimentary phrase, "fishing expeditions." While cDNA produced from tumours is
hybridised to a chip to examine gene expression levels, fluorescently labelled DNA from
tumour samples can be used to measure changes in DNA copy number (gene amplification or
deletion).TMAs are created by inserting tissue cores into holes that have already been cored
in a recipient paraffin block.

APPLICATIONS –
Microarray technology has a wide range of applications in various fields of biology and
medicine. Here are some examples of how microarrays are used:

1. Gene expression profiling: Microarrays can be used to analyze the expression of

thousands of genes simultaneously in a single experiment. This can be used to identify
genes that are differentially expressed in different tissues, diseases, or under different
conditions.

2. Genotyping and SNP analysis: Microarrays can be used to genotype individuals by

detecting single nucleotide polymorphisms (SNPs) across the genome. This is useful
for identifying genetic variations that are associated with diseases or for mapping the
genetic basis of complex traits.
3. Comparative genomic hybridization (CGH): CGH is a technique that uses microarrays
to detect copy number variations (CNVs) in the genome. CNVs are changes in the
number of copies of a particular DNA sequence, and can be associated with diseases
such as cancer.

4. ChIP-chip analysis: Chromatin immunoprecipitation (ChIP) combined with

microarrays can be used to identify the DNA sequences that are bound by specific
proteins, such as transcription factors. This can help to understand gene regulation and
identify potential drug targets.
5. Protein microarrays: Microarrays can be used to study protein-protein interactions,
identify protein binding partners, and screen for drug candidates.
Overall, microarrays have revolutionized the field of genomics and have opened up new
avenues for research in various fields of biology and medicine.

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CONCLUSION –
This review has given a small outline of the technique behind microarray and the various
steps involved. The technique, though limited at present in its applications due to the cost
factor, may widen its prospects once there is increase in the availability of commercial
products. The ability to record large number of old samples and analyzing them for various
genetic alterations helps in understanding the concept of molecular biology. Microarrays hold
much promise for the analysis of diseases in the oral cavity. Classifications of oral disease by
DNA, RNA, or protein profiles will greatly enhance our ability to diagnose, prevent, monitor
and treat our patients. Currently, microarrays are primarily a research tool. Microarrays
promise a more biologically based, individualized, and vastly improved standard for oral
care, which will have great clinical impact on the way dentistry will be practiced in the
future.