DNA Microarray Technology

(DNA Chip Technology)

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University of Management and Technology

Submitted to: Mr. Muhammad Khurram

Lab Course: Biochemistry 1

Section: A2
DNA Microarray Technology
In Molecular Biology, there is a vast variety of processes and techniques evolved
through the development of the technologies being born now-a-days. Though these
processes are accurate enough but, these are not suitable for studying a large number
of genes. DNA Microarray Technology is such a technology which is enabling
scientists to explore and understand issues which were once unrecognizable. An
Array is the arrangements of samplexs in a particular order where we match known
and unknown DNA samples based on the pairing rules. It helped the researchers to
understand the elementary aspects of growth and development of life, exploring the
genetic causes of diseases and tracing the evolutuionary histories of organisms.
DNA Microarray commonly known as a DNA chip or Biochip is a group of
microscopic fragments of DNA attached to a solid
surface. Each DNA spot is as small as 1 picomole
(10 -12 moles) and a small chip has thousands of spots
on it. Originally, DNA chips were about 9 cm ×
12 cm called Macroarrays because initially they
were of so large size but now, they are more
portable and smaller about the size of a millimeter
or so.
The property of complementary nucleic acid In the following micrograph, hybridized
sequences specifically pair up with each other by DNA sequences also called anti -sense
forming Hydrogen bonds between complementary RNA is att ached to a silicon chip. The
base pairs in a nucleotide sequence which means fi rst computerized analysis was pictured
in 1981.
tighter non-covalent interaction in two strands of
DNA. Only strongly paired strands remains
hybridized and the non-specific bonds break lately. Flouroscently labeled probes or
target sequences binds to a probe sequence generating a signal depending upon the
hybridizing conditions like temperature and washing after hyubridization. Microarrays
uses Relative Quantitation in which the intensity of a feature is being compared to
the intensity of the other feature in a different condition to evaluate the effect of a
variable on the genome which is further known by its position.
In the previous years, MAT is extensively used by the scientists and over the years it is becoming
more and more important. Over the years, a lot of generation of data related to the expression of
genes is collected. But, the data is scattered over the world and is not readily available. For this
purpose, organizations such as National Center for Biotechnology Information (NCBI) has
formulated Gene Expression Omnibus or GEO which gives vast information oj vairied sources
being analyzed by MAT.
In a Typical Microarray Experimemt, the DNA template synthizes a mRNA
molecule. When many DNA samples are collected, they are used to form an
“ARRAY”. The amount of the mRNA bounded onto each side gives the expression
level of many genes with number running n thousands upto ten thousands. Data is
composed and a profile is generated to illustrate the gene expression in the cell.
Microarray Technique uses common assay systems like microplates or standard
bottling membranes. The size of sample spot is typically upto 200 microns or less in
diameter but it contains thousands of such spots. These thousands of spotted samples
are called Probes. Probes have known identity and are immobilized onto a solid
support which includes a microscope glass slide, silicon chips or nylon membrane.
Scientists found that the spots can be DNA, oligonucleotides or cDNA. An order of
arrangements is very important because the location of each probe is specific on the
chip which gives similarity indices of two samples to be compared. These are used in
determining complementary binding of unknown sequences hence allowing parallel
analysis of gene expression and gene sequences. An experiment with only a single
chip is capable of storing information of thousands of genes simultaneously.
Depending upon the kind of an immobilized sample, constructed arrays and the
information obtained, 3 types of microarrays are used
in common
1. Microarray Expression Analysis uses the
cDNA derived from single-stranded mRNA or
miRNA (micro RNA). A suitable enzyme known as
REVERSE TRANSCRIPTASE is used in the
synthesis. Thus, this technique is allowing the
researchers to express that genes which are not
normally expressed known as Hetrologous
expression. Therefore, it can be used to analyze the
transcriptomic profiles in bulks of tissues, cells or
even single nuclei. In bioinformatics, the term cDNA is used to express DNA bases
in the form of deoxy-CGAT instead of CGAU. Figure shows an output of
microarray of cDNA being used in testing of genes. The immobilized cDNA
sample has both the genes for diseased as well as normal expressions. But the spots
with more intensity are comparising the diseased genes if the gene is over-
expressed in a certain condition. For example overexpression of MYC, REL or
HER2 genes often causes a diversity of human body cancers. Overexpression of
some genes like CAPN10 is related with diabetes mellitus type 2 in which body
cells become resistant to insulin and cannot take enough glucose. So that glucose
remains into the blood.
2. Mutation analyzing Microarrays uses gDNA when a gene is different from
each other by even a single nucleotide base. gDNA is basically genomic DNA. The
genome is the heridity information centre of an organism which is the centre of
formation of mRNA containing 3-nucleotide sequences called codons which are
then used to form long amino-acid chains called polypeptides. A single
replacement of a nucleotide can change the codons so, changes the normal protein
structure. So,this technique compares genomes which have just a replacement of a
single nucleotide. A Single Nucleotide Polymorphism are gene samples differing
by only 1 nucleotide sequence. Their method of detection is known as SNP
detection. This method is being used to detect point mutations.
3. Comparative Genomic Hybridization is basically a molecular Cytogenic
method to analyze duplicated or copied number variatiobns (CNVs). It is used to
identify the increase or decrease of chromosomal fragments being tested or
 analyzed to harbour genes involved in a disease. This technique was originally
aimed to compare chromosomal complements of tumor Vs normal tissues. The two
different samples form different sources are labelled with flouroscent dyes usually
red and green. The higher intensity of a single colour gives the overproduction of a
certain protein or molecule while the lower intensity of colour gives the
information that the cause of tumor is basically the loss of a gene component. The
flouroscent dyes are mostly red and green in colour. CGH is not a very reliable
method it has some drawbacks too. It is only suitable for detection of
Chromosomal Abbressions such as inversion, duplication, reciprocal
translocations or ring chromosomes. This process renders some limitations as it
only gives account for chromosomal abbressions with copy number changes. The
other chromosomal abbressions like mosaicism, balanced chromosomal
translocations (translocation of genes with same no. of nucleotides) and
inversions. These mutations donot posesses gains or losses relative to ploidy level
so, cannot be detected. Before CGH, the repititive DNA sequences (centromeres
and Telomeres) must be blocked to obtain accurate results otherwise it can render
them duplications.
However, it was used in Human Genome sequencing of all 46 chromosomes
known as genomic map. This technique can be of two types of which Array CGH
has overcame many of the limitations of CGH:
 Conventional CGH uses a reference metaphase spread as the target.
Abbressions smaller than 5-10 Mb cannot be detected by this method due
to low resolution of metaphase chromosomes
 In Array CGH the targets are genomic fragments which are cloned in a
variety of vectors like BACs or plasmids, cDNAs or may be
oligonucleotides. Equal quantities of microarray fragments are mixed and
cohybridized and triplicated on the array. If two portions of DNA chip had
equal ratio of copies, then this region oif patient’s DNA is normal. Altered
ratios for example 3:5 or 6:2 indicates the loss or gain of DNA at a
specific genomic region thus analyzing the Duplication mutations.
Microarray has a vast variety of Applications in Molecular Biology and Bioinformatics. Some
of them are listed below:
 In Gene Discovery, DNA Microarray Technology helped in identifying new genes,
knowing about their functions and expression levels under different conditions. Human
Genome Seuquencing used these techniques to formulate the genomic map of all 46
chromosomes of humans, drosophila, grasshoppers, bacteria, viruses like T2 and T4
phages and many other organisms.
 DMT helped researchers in diagnosis of diseases such as heart diseases, mental illnesses,
infectious ailments like cancers and tumors. Before the development of DMT, types of
cancers were classified on the basis of the type of organs in which the tumor develops.
Now, researchers are classifying tumors on the basis of patterns of genes and their
activity in the tumor cells. This tremendously targeted directly the genetic causes of
tumor to evaluate a specific type of cancer.
 Discoveries of Drugs by microarray technology in pharmaceutics, pharmacogenomics
and pharma industries is very common. Pharmacogenomics is the study of relations
and correlations between the genomic profile of a patient and apeutic responses.
Comparative analysis technique of the genes from a diseased to a normal cell is being
used. The researchers used the information for the synthesis of drugs to combat with such
proteins and reducing their effects.
 MAT is used in Toxicology Research providing robust platforms for the research of the
effects of toxins on the cell and their passage into the progeny. The branch of molecular
biology which relates toxin study with the changes in the genetic profiles of
chromosomes in the cell when it is exposed to such toxins is called Toxicogenomics.
 SNP arrays for polymorphism is an application of MAT used in diseases caused by
pathogens and GWAS analysis. GWAS analysis is also called as Genome-wide
Association study. It is the observational study of a genome-wide set of genetic variants
in the individuals to see whether any variant is associated with a particular trait. GWAS
focuses on SNP arrays and traits like major human diseases like cancer, hear diseases,
diabetes etc.
 MAT is also used in the identification od structrural variations of proteins and the
measurement of gene expression. For example, in Cystic Fibrosis, a Cl- transmembrane
carrier protein is made faulty due to some point mutations in human body.

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