Download Document

Microarray
Technology
Asma-ul-husna
07-arid-1237
Ph.D (Zoology)
1st Semester
Microarray
Microarray is a tool for analyzing gene expression that
consists of a small membrane or glass slide
containing samples of many genes arranged in a
regular pattern.”
History
• Microarray technology evolved from Southern blotting, where
fragmented DNA is attached to a substrate and then probed
with a known DNA sequence.
• The use of miniaturized microarrays for gene expression
profiling was first reported in 1995, and a complete eukaryotic
genome (Saccharomyces cerevisiae) on a microarray was published
in 1997.
Introduction
• The underlying principle of microarray technology is
the ability of DNA to bind to itself and to RNA.
• Analyzing gene expression involves the detection of
mRNA species (transcriptome) present in a cell or
tissue at a particular point in time.
Principle
•
DNA microarrays have provided a new and powerful tool to
perform important molecular biology and clinical diagnostic assays.
•
The basic idea behind DNA microarray technology has been to
immobilize known DNA sequences referred to as probes in
micrometer-sized spots on a solid surface (microarray) and
specifically hybridize a complementary sequence of the analyte DNA
or a target.
•
A fluorescently labeled reporter facilitates fluorescent detection of
the presence or absence of a particular target or gene in the
sample.
•
By using laser-scanning and fluorescence detection devices such as
CCD cameras, different target hybridization patterns can be read
on the microarray and the results quantitatively analyzed.
The plate
• Usually made commercially.
• Made of glass, silicon, or nylon.
• Each plate contains thousands of spots, and each
spot contains a probe for a different gene.
• A probe can be a cDNA fragment or a synthetic
oligonucleotide, such as BAC (bacterial artificial
chromosome set).
• Probes can either be attached by robotic means,
where a needle applies the cDNA to the plate, or
by a method similar to making silicon chips for
computers. The latter is called a Gene Chip.
Procedure
1) Collect Samples.
2) Isolate mRNA.
3) Create Labelled DNA.
4) Hybridization.
5) Microarray Scanner.
6) Analyze Data
1: Collect Samples
• This can be from a variety of organisms. We’ll use two
samples – cancerous human skin tissue & healthy
human skin tissue
2: Isolate mRNA.
• Extract the RNA from the samples. Using either
a column, or a solvent such as phenolchloroform.
• After isolating the RNA, we need to isolate the
mRNA from the rRNA and tRNA. mRNA has a
poly-A tail, so we can use a column containing
beads with poly-T tails to bind the mRNA.
• Rinse with buffer to release the mRNA from the
beads. The buffer disrupts the pH, disrupting
the hybrid bonds.
3: Create Labelled DNA

Add a labelling mix to the RNA.
The labelling mix contains polyT (oligo dT) primers, reverse
transcriptase (to make cDNA),
and fluorescently dyed
nucleotides.

We will add cyanine 3
(fluoresces green) to the healthy
cells and cyanine 5 (fluoresces
red) to the cancerous cells.

The primer and RT bind to the
mRNA first, then add the
fluorescently dyed nucleotides,
creating a complementary
strand of DNA
4: Hybridization
•
Apply the cDNA we
have just created to a
microarray plate.
•
When comparing two
samples, apply both
samples to the same
plate.
•
The ssDNA will bind to
the cDNA already
present on the plate.
5: Microarray Scanner

The scanner has a laser, a
computer, and a camera.

The laser causes the hybrid
bonds to fluoresce.

The camera records the images
produced when the laser scans
the plate.

The computer allows us to
immediately view our results
and it also stores our data.
6: Analyze the Data

GREEN – the healthy sample hybridized more
than the diseased sample.

RED – the diseased/cancerous sample hybridized
more than the nondiseased sample.

YELLOW - both samples hybridized equally to
the target DNA.

BLACK - areas where neither sample hybridized
to the target DNA.

By comparing the differences in gene expression
between the two samples, we can understand more
about the genomics of a disease.
6. Continued
Biological Samples in 2D Arrays on Membranes
or Glass Slides
Whole process through
animation
• http://highered.mcgrawhill.com/olcweb/cgi/
pluginpop.cgi?it=swf::535::535::/sites/dl/free
/0072437316/120078/micro50.swf::Micr
oarray
• http://www.digizyme.com/competition/exa
mples/genechip.html
Types of microarrays
It include:
•
DNA microarrays,
•
oligonucleotide microarrays
•
Protein microarrays
•
Tissue microarrays
•
Cellular microarrays (also called transfection microarrays)
•
Chemical compound microarrays
•
Antibody microarrays
•
Carbohydrate arrays (glycoarrays)
Design of a DNA microarrays
The principle of DNA microarrays lies
on the hybridization between the
nucleotide. Using this technology the
presence of one genomic or cDNA
sequence in 1,00,000 or more
sequences can be screened in a single
hybridization.
The property of complementary
nucleic acid sequences is to
specifically pair with each other by
forming hydrogen bonds between
complementary nucleotide base pairs.
Oligonucleotide Microarray
Protein Microarray
Tissue & Antibody Microarray
TRANSFECTION MICROARRAY
(A) Outline of the procedure (B) An example of the transfection
microarray format. HEK 293 cell line was transfected.
Chemical compound microarray
Carbohydrate Microarray
Applications
The DNA chips are used in many areas as given below:
•
Gene expression profiling
•
Discovery of drugs
•
Diagnostics and genetic engineering
•
Alternative splicing detection
•
Proteomics
•
Functional genomics
•
DNA sequencing
•
Toxicological research (Toxicogenomics)
ADVANTAGES
•
Provides data for thousands of genes.
•
One experiment instead of many.
•
Fast and easy to obtain results.
•
Huge step closer to discovering cures for diseases and cancer.
•
Different parts of DNA can be used to study gene expression.
Disadvantages
•
The biggest disadvantage of DNA chips is that they are expensive to
create.
•
The production of too many results at a time requires long time for
analysis, which is quite complex in nature.
•
The DNA chips do not have very long shelf life, which proves to be
another major disadvantage of the technology.
Summary
•
DNA Microarrays are one of the most effective invention ever
developed.
•
A DNA Microarray is a test that allows for the comparison of
thousands of genes at once.
•
Microarray technology uses chips with attached DNA sequences as
probes for gene expression.
•
Any DNA in the sample that is complementary to a probe sequence
will become bound to the chip.
•
Microarray technology is most powerful when it used on species
with a sequenced genome. The microarray chip can hold sequences
from every gene in the entire genome and the expression of every
gene can be studied simultaneously.
•
Gene expression data can provide information on the function of
previously uncharacterized genes.
Thank you for
your patience
??????????