DNA Microarray and its application in

Cancer diagnosis

By
Brijesh Singh Yadav
Division of Biochemistry
Indian Veterinary Research institute
Izatnagar,India.243122
 What we will be discussing…

• What is DNA microarray?

• The purpose of using DNA microarray
• Oligonucleotide Probes
• The plate.
• Steps to perform a microarray.
• Application in Cancer Biology.
• Future perspectives.
 What is DNA Microarray?
• Scientists used to be able to perform genetic analysis of a
few genes at once. DNA microarray allows us to analyze
thousands of genes in one experiment!
• It consists of an arrayed series of thousands of microscopic
spots of DNA oligonucleotides, each containing a specific DNA
sequence, known as probes.
 Purposes
• So why do we use DNA microarray?
– To measure changes in gene expression levels – two
samples’ gene expression can be compared from
different samples, such as from cells of different stages
of mitosis.
– To observe genomic gains and losses. Microarray
Comparative Genomic Hybridization (CGH)
– To observe mutations in DNA.
 Oligonucleotide Probes

 A piece of DNA that binds specifically

to a particular gene or part of a
gene.
• The sequence of the gene probe must be
complementary to the nucleotide bases of
the specific mRNA or DNA sequence of
interest.
• Gene probes can be as small as 20-40
base pairs or be up to 1000bp long.
 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.
• 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.
Let’s perform a microarray!
1) Probes Designing
2) Collect Samples.
3) Isolate mRNA.
4) Create Labelled DNA.
5) Hybridization.
6) Microarray Scanner.
7) Analyze Data.
 STEP 1: Probes Designing

• DNA probes should be designed considering the

various aspect such as melting temperature
(Tm),guanine /cytonine (G/C)content, secondary
structure, length and binding position within the
target DNAs
• These factor can effect the signal intensity,
specificity and sensitivity following hybridization.

• Longer probes are more specific to individual

target genes, and shorter probes may be spotted
across the array in higher densities and are
cheaper to manufacture.
 STEP 2: Isolate mRNA.
• Extract the RNA from the samples. Using either a column,
or a solvent such as phenol-chloroform.

• 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.
 STEP 3a: Collect Samples

 This can be from a variety of organisms. We’ll use two

samples – cancerous human skin tissue & healthy
human skin tissue
STEP 3b: Create Labelled
DNA.
 Add a labelling mix to the
RNA. The labelling mix
contains poly-T (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
STEP 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.
STEP 5a: LASERS!
 STEP 5b: 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.
 STEP 6: Analyze the
Data.
 GREEN – the healthy sample
hybridized more than the diseased
sample.

 RED – the diseased/cancerous sample

hybridized more than the non
diseased 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.
Application in Cancer Biology
 Cancer
• CANCER is a genetic disorder, mostly resulting
from acquired mutations and epigenetic changes
that influence gene expression.
• The use of DNA microarrays for the
comprehensive analysis of RNA expression
(expression profiling) in human tumor sample
should much promise.
 Electron Microscope Photographs of Cancer Cells

Breast Cancer Cell Brain Cancer Cell 

Cancer cell being attacked by the immune system Prostate cancer cell
 Microarray in Cancer

• The advent of the technology of DNA microarrays

constitutes an epochal change in the classification
and discovery of different types of cancer because
the information provided by DNA microarrays
allows an approach to the problem of cancer
analysis from a quantitative rather than qualitative
point of view.
• Cancer classification requires well founded
mathematical methods which are able to predict
the status of new specimens with high significance
levels starting from a limited number of data.
 Working with cancer
• The mRNA is a copy of the DNA and, therefore, is
complimentary to the DNA it was copied from.
• mRNA is placed onto the chip, the mRNA that was
expressed from the DNA will bind onto the DNA
that it is complementary.
• As a result, the mRNA that binds with the DNA on
the chip will give off a fluorescent dye that can be
analyzed by a computer. The brighter the color
that is present on the computer screen, the more
active the gene is and the less color, or even no
color, means that a gene is not being expressed.
 DNA Microarray
samplle-1 samplle-2
chromosome

PCR Purification of mRNA

Gene A Gene C
Gene B
Labeling during RT
Arrayer
Glass
Slide Gene A Gene B Gene C

(Target)
(Probe)
16 hr, 42C Wash

Microarray
Gene A Gene B Gene C
Scan

Image Analysis: samplle-2 samplle-1,2 samplle-1

 Cancer Data Analysis

Microarray Pearson's correlation coefficient

Spearman's correlation coefficient
Euclidean distance
Cosine coefficient
Expression data Information gain
Mutual information
Signal to noise ratio

Feature Selection
3-layered MLP with back propagation
Cancer Predictor k-nearest neighbor
Support vector machine
Structure adaptive self-organizing
map
Tumor Normal Ensemble classifier

Cancer classification system

 The Future of DNA Microarray
• Gene discovery. DNA Microarray technology helps in the
identification of new genes, know about their functioning
and expression levels .
• Disease diagnosis. classify the types of cancer on the
basis of the patterns of gene activity in the tumor cells.
• Pharmacogenomics. is the study of correlations between
therapeutic responses to drugs and the genetic profiles of
the patients.
• Toxicogenomics. microarray technology allows us to
research the impact of toxins on cells. Some toxins can
change the genetic profiles of cells, which can be passed on
to cell progeny.
 Personalized Medicine
• Expression patterns and
levels, referred to as
the brightness from the
fluorescent dye, allows
scientists to be able to
determine which genes
are being active in a
cancerous cell.
• Personalized treatment
plans can be developed
for each individual
patient.
 Sources…..
 DNA Microarray Technology. National Human Genome Research Institute, 17
Dec. 2009. 19 Feb. 2010 <http://www.genome.gov/10000533>

 Microarrays: Chipping Away at the Mysteries of Science and Medicine.

National Center for Biotechnology Information, 27 July 2007. 19 Feb. 2010.
<http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html>

 Brown, P.O. & Botstein, D. Exploring the New World of the Genome with DNA
Microarrays. Nature Genetics Supplement. 21. (1999): 33-37. <
http://www.ctu.edu.vn/~dvxe/Bioinformatic/PDF%20Files/Volume21/ng0199s
upp_33.pdf
>

 Simon, R., Radmacher, M.D., Dobbin, K., & McShane, L.M. Pitfalls in the Use
of DNA Microarray Data for Diagnostic and Prognostic Classification. Journal of
the National Cancer Institute. 95. (2003): 14-18.
http://jnci.oxfordjournals.org/cgi/content/full/95/1/14

 Holloway, A.J., Van Laar, R.K., Tothill, R.W., & Bowtell, D.D.L. Options
Available – From Start to Finish – For Obtaining Data From DNA Microarrays II.
Nature Genetics Supplement. 32. (2002): 482-489.
<http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/ng1030.pdf>
 Special Thanks to…………
• Dr.Bhaskar Sharma(ICAR, National Professor, IVRI)
• Dr.K.P.Mishra (VC, Nehru Gram Bharti University )
• Dr.Ajay Kumar(Scientist, Division of Biotechnology, IVRI)
• Dr.Meeta Saxena(Scientist, Division of Biochemistry,IVRI)
• Mr.Mayank Pokhriyal (RA, Division of Biochemistry,IVRI)
• Dr.Barkha Ratta (RA, Division of Biochemistry,IVRI)
• Mr.Gaurav Rai (SRF, Division of Biochemistry,IVRI)
Send your suggetion to me at my email:-brijeshbioinfo@gmail.com