How microarray technology works?
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Introduction to Microarray:
Molecular Biology research
evolves through the development of the
technologies used for carrying them
out. It is not possible to research
on a large number of genes using traditional
methods. DNA Microarray is one such technology
which enables the researchers to investigate
and address issues which were once thought
to be non traceable. One can analyze
the expression of many genes in a single
reaction quickly and in an efficient
manner. DNA Microarray technology has empowered
the scientific community to understand
the fundamental aspects underlining
the growth and development of life as
well as to explore the genetic causes
of anomalies occurring in the functioning
of the human body.
A typical microarray experiment
involves the hybridization of an mRNA
molecule to the the DNA template from
which it is originated. Many DNA samples
are used to construct an array. The
amount of mRNA bound to each site on
the array indicates the expression level
of the various genes. This number may
run in thousands. All the data is collected
and a profile is generated for gene
expression in the cell.
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Microarray
Technique
An array is an orderly
arrangement of samples where matching
of known and unknown DNA samples is
done based on base pairing rules. An
array experiment makes use of common
assay systems such as microplates or
standard blotting membranes. The sample
spot sizes are typically less than 200
microns in diameter usually contain
thousands of spots.
Thousands of spotted samples known as
probes (with known identity) are immobilized
on a solid support (a microscope glass
slides or silicon chips or nylon membrane).
The spots can be DNA, cDNA, or oligonucleotides.
These are used to determine complementary
binding of the unknown sequences thus
allowing parallel analysis for gene
expression and gene discovery. An experiment
with a single DNA chip can provide information
on thousands of genes simultaneously.
An orderly arrangement of the probes
on the support is important as the location
of each spot on the array is used for
the identification of a gene. |
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Types
of Microarrays:
Depending upon the kind
of immobilized sample used construct
arrays and the information fetched,
the Microarray experiments can be categorized
in three ways:
1. Microarray expression analysis: In this experimental setup, the
cDNA derived from the mRNA of known
genes is immobilized. The sample has
genes from both the normal as well as
the diseased tissues. Spots with more
more intensity are obtained for diseased
tissue gene if the gene is over expressed
in the diseased condition. This expression
pattern is then compared to the expression
pattern of a gene responsible for a
disease.
2. Microarray for mutation analysis: For this analysis, the researchers
use gDNA. The genes might differ from
each other by as less as a single nucleotide
base. A single base difference between
two sequences is known as Single Nucleotide
Polymorphism (SNP) and detecting them is known as SNP detection
3. Comparative Genomic Hybridization: It is used for the identification
in the increase or decrease of the important
chromosomal fragments harboring genes
involved in a disease. |
Applications
of Microarrays:
Gene discovery: DNA Microarray
technology helps in the identification
of new genes, know about their functioning
and expression levels under different
conditions.
Disease diagnosis: DNA Microarray technology helps researchers
learn more about different diseases
such as heart diseases, mental illness,
infectious disease and especially the
study of cancer. Until recently, different
types of cancer have been classified
on the basis of the organs in which
the tumors develop. Now, with the evolution
of microarray technology, it will be
possible for the researchers to further
classify the types of cancer on the
basis of the patterns of gene activity
in the tumor cells. This will tremendously
help the pharmaceutical community to
develop more effective drugs as the
treatment strategies will be targeted
directly to the specific type of cancer.
Drug discovery: Microarray
technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations
between therapeutic responses to drugs
and the genetic profiles of the patients.
Comparative analysis of the genes from
a diseased and a normal cell will help
the identification of the biochemical
constitution of the proteins synthesized
by the diseased genes. The researchers
can use this information to synthesize
drugs which combat with these proteins
and reduce their effect.
Toxicological research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation
between responses to toxicants and the
changes in the genetic profiles of the
cells exposed to such toxicants.
GEO:
In the recent past, microarray technology
has been extensively used by the scientific
community. Consequently, over the years,
there has been a lot of generation of
data related to gene expression. This
data is scattered and is not easily
available for public use. For easing
the accessibility to this data, the National Center for Biotechnology
Information (NCBI) has formulated
the Gene Expression Omnibus or GEO. It is a data repository
facility which includes data on gene
expression from varied sources.
Microarray
probe design parameters:
For 25-35 mers
Parameter |
Minimum
Value |
Maximum
Value |
Default
Value |
Unit |
Probe
Length |
10 |
99 |
30 |
bases |
Probe
Length tolerance |
0 |
15 |
3 |
|
Probe
Target Tm |
40 |
99 |
63 |
°C |
Probe
Tm Tolerance (+) |
0.1 |
99 |
5 |
|
Hairpin
Max ÄG |
0.1 |
99.9 |
4 |
Kcal/mol |
Self
Dimer ÄG |
0.1 |
99.9 |
7 |
Kcal/mol |
Run/Repeat |
2 |
99 |
4 |
bases |
For 35-45 mers
Parameter |
Minimum
Value |
Maximum
Value |
Default
Value |
Unit |
Probe Length |
10 |
99 |
40 |
bases |
Probe Length
tolerance |
0 |
15 |
3 |
|
Probe Target
Tm |
40 |
99 |
70 |
°C |
Probe Tm Tolerance
(+) |
0.1 |
99 |
5 |
|
Hairpin Max
ÄG |
0.1 |
99.9 |
6 |
Kcal/mol |
Self Dimer ÄG |
"
0.1 |
99.9 |
8 |
Kcal/mol |
Run/Repeat |
2 |
99 |
5 |
bases |
For 65-75 mers
Parameter |
Minimum
Value |
Maximum
Value |
Default
Value |
Unit |
Probe Length |
10 |
99 |
70 |
bases |
Probe Length
tolerance |
0 |
15 |
3 |
|
Probe Target
Tm |
40 |
99 |
75 |
°C |
Probe Tm Tolerance
(+/- above ) |
0.1 |
99 |
5 |
|
Hairpin Max
ÄG |
0.1 |
99.9 |
6 |
Kcal/mol |
Self Dimer ÄG |
0.1 |
99.9 |
8 |
Kcal/mol |
Run/Repeat |
2 |
99 |
6 |
bases |
Other Parameters
DNA
Microarray with Array Designer:
Array Designer is an exceptional software
to design highly specific oligos for expression and SNP genotyping microarray experiments.
References:
1.Validation of Sequence-Optimized
70-base Oligonucleotides for Use on
DNA Microarrays (http://www.westburg.nl/download/arrayposter.pdf). By John Ten Bosch, Chris Seidel,
Sajeev Batra, Hugh Lam, Nico Tuason,
Sepp Saljoughi, and Robert Saul.
2. Assessment
of the specificity and Sensitivity of
the oligonucleotides (50 mer ) microarrays. By Dr. Susanne Schröder1,
Dr. Jaqueline Weber2, and Dr. Hubert
Paul 1 WG Biotech AG, Microarray Development1
and Department of Bioinformatics2.
3. 50
nucleotide long probes on microarrays
enable high signal intensity and high
specificity. By Dr. Susanne
Schröder1, Dr. Jaqueline Weber2,
and Dr. Hubert Paul1 MWG Biotech AG,microarray
Development1and Department of Bioinformatics
2,
Anzinger Str. 7, 85560 Ebersberg, Germany.
4.Optimization
of Oligonucleotide - based DNA microarrays. By Angela Relogio, Christian
Aschwager, Alexandra Ritcher, Wilhelm
Ansorge and Juan Valcarel.
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