CYTOGENETICS LEC

BSMT 2 - 2ND SEMESTER

DNA MICROARRAY

Lesson 10: DNA Microarray

Mrs. Jamie Go

GENETICS VS. GENOMICS

GENETICS GENOMICS
● Study of heredity ● Study of genes, how they
○ Genes passed on from interact and expressed as a
generations to generations whole
○ On how they interact, and
● It focuses primarily on the
how they are passed on
likelihood of developing from generation and
cancer expressed as a whole
● Genetics tests finds mutations, ● Genetics and Genomics are
not diseases profiling tools that focuses on
cancer itself and can help
determine:
1. How aggressive the cancer
is (prognosis)?
2. What is the likely benefit
from the treatment
(prediction)?
a. If the treatment is ● DNA + histones forms nucleosomes
effective towards the ● Summary:
cancer DNA → DNA + histones → Nucleosomes → Chromatin
fiber → Chromatin → Duplicated chromosome
HUMAN GENOMICS PROJECT ● Part of chromosome:
● As part of genomics ○ Telomere
● It is an international scientific research project ○ Centromere
○ Determining the sequence of human DNA ○ Chromatid
○ Identifying and mapping all of the human genes ● Each human cell contains the 46 chromosomes and each
● Key findings: chromosome contains 20,500 genes.
○ There are approximately 20,500 genes in a human being ● Chromosome is a condensation of DNA.

GENES STRUCTURAL GENES VS. REGULATORY GENES

● They are a part of a DNA STRUCTURAL GENES REGULATORY GENES
● They are the basic physical and functional unit of heredity A gene that codes for any RNA or A gene involved in controlling the
● Genes are made up of DNA. Some genes act as instructions to protein product other than a expression of one or more other
make molecules called proteins regulatory factor genes
● The nucleotide sequence is (A-T, C-G) on the DNA Encode for proteins or RNA Encode for transcription factors
or regulatory RNA
Encode for mRNAs, rRNAs, and Encode for regulatory RNA such
tRNAS as miRNAs and siRNAs
Gene products have either Regulate the expression of
structural or functional structural genes.
importance
Regulatory genes process the
turning on and off of the genes.
E.g.They are the ones that
decide when to activate the
acting gene to produce protein
Regulatory genes ensure that the
appropriate genes are expressed
at the proper time.
Examples: Examples:
● Lac Z, lac Y, and lac A ● Lac I gene, CAP gene,
genes of the lac operon, etc.
actin gene, etc.

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 WHOLE DNA/GENES cancer allows the further diagnosis, progression
and aggressiveness analyses, prognosis, and
prediction of therapeutic treatment.

MAIN TYPES OF GENE EXPRESSION ASSAYS

● Serial Analysis of Gene Expression (SAGE)
● Short Oligonucleotide Arrays (Affymetrix)
○ It does not use the whole sequence of genes. It only uses
a small part sequence of the gene.
● Long Oligonucleotide Arrays (Agilent Inkjet)
● Fiber Optic Arrays (Ilumina)
● cDNA Arrays (Brown/Botstein)
● Whole DNA/Gene is divided into two parts: ○ This is a slide cDNA Microarray which is discovered by
1. Intron ● Portion of a gene that does not code for amino Brown and Botstein.
acids/proteins ○ Each slide contains multiples spot and each spot
○ Like intermission, they are there para naay contains a DNA clone.
time mu form ug final mature RNA ang ○ Usually, PCR generated are samples for cDNA Arrays
whole DNA.
● It will later then be removed by RNA Splicing HISTORICAL BACKGROUND OF DNA MICROARRAY
● In between 2 exons is 1 intron
2. Exon ● Portion of a gene that will form a part of the final Mark Schena Father of Microarray Technology
mature RNA produced by that gene after introns Steve Fooder Developed a scanner for reading the output
have been removed by RNA splicing.
Patrick Brown and Quantitative monitoring of Gene Expression
GENE EXPRESSION Mark Schena Patterns with complementary DNA Microarray

● DNA Microarray is based on the Principle of Southern Blotting

in the year 1975.
● The concept of DNA Microarray began in the mid 1980s.
● Also commonly known as “DNA Chip” or “biochip”
● It is composed of a collection of microscopic DNA steps
attached to a solid surface.
● It is a technology that can sequence thousand of genes at a
time in order to quantify the expression of the said genes.
● Microarray technology uses a microarray chip which contains
Normal cell is compared to a cancer cell. probes at different locations on its surface that can bond to
A normal cell has only Gene A and C are being activated while for the target DNA.
Cancer Cell are Gene B and C are activated. If maactivate ang Gene B it ● The bonding of probes and targets is referred to as
means that naay cancer ang patient. So, inig after sa gene expression, “Hybridization”
once maactivated ang Gene B that is indication of Cancer. ● Microarrays can be used to study gene expressions in different
conditions or in different cells
● Gene expression occurs only in a particular type of cell or
● Target DNA - this is the DNA of a sample of interest which is
tissue where only a subset of an organism’s DNA will be
compared to a reference DNA or “control DNA”
expressed as mRNAs at any given time
● The differential binding and expression of fluorescent probes
○ Central Dome of life is from the DNA to the RNA to protein.
on the chip allows the user to determine the relative expression of
○ Focuses on the Central Dome of life
target genes
○ Where 20,500 genes are activated simultaneously and these
● Relies on the hybridization properties of nucleic acids to
genes are only activated when they already need it.
monitor DNA or RNA abundance on a genomic scale in different
■ Suppose after eating food, our body needs insulin,
types of cells.
the genes in our pancreas gets activated then they
send mRNA to activate the code for protein PRINCIPLE OF DNA MICROARRAY
synthesis.
● The core principle behind microarrays is the hybridization
■ From DNA musend sa message sa mRNA na
between 2 DNA strands where fluorescent labeled cDNA or
muproduce ug insulin. After maactivate,
nucleotide (target) binds to a DNA on the slide (probe), will
makaproduce na ug insulin ato pancreas.
then generate a signal that depends on the strength of the
● Proteins do most of the work
hybridization determined by the number of paired bases.
○ Proteins are either dynamically created or destroyed
○ The gene that is placed into the whole in the glass slide is
○ mRNA blueprints are also dynamically created or destroyed
called a probe.
○ Different mRNAs expressed at different times/places
○ Microarrays in general are a snapshot that captures the
○ Important to know the mRNA expression levels
activity patterns of thousands of genes at once and
● The unique pattern of gene expression for a given cell or tissue is
microarrays also allow simultaneous measurement of
referred to as its molecular signature.
the level of the transcription of every gene in a
○ Why is gene expression important in any
genome. This is what we consider as a gene expression.
malignancy/diseases?
■ The use of gene expression profiling and
development of gene biomarkers or signatures for

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 TWO (2) COMMON METHODS OF DNA MICROARRAY
THE ARRAY/SLIDE
1. Glass Slide cDNA Microarray

● A collection of features spatially arranged in a two-dimensional

grid, arranged in columns and rows
● The slide consists of a thousand to hundreds of thousands of
● Conventional method
spots per square inch
● First type of DNA Microarray technology developed
● Each spot holds millions of copies of a DNA sequence from one
● Invented by Patrick Brown & his colleagues at Stanford
gene
University
● An array is composed of multiple tiny dot (in the picture)
● Uses a robotic device which deposits (spots) a nanoliter sample
● Each dot contains one complimentary copy or one single probe of
of DNA onto a coated microscopic glass slide (50-150
DNA
micrometer in diameter)
○ Mao ni siya e use for conventional slide method.
● The slide is usually made commercially, and is made of
3 MAIN TYPES OF DNA MICROARRAY glass/silicon/nylon.
● Each slide contains thousands of spots, and each spot contains a
● These arrays use a poly-lysine coated
probe for a different gene.
microscope slide
● A robotic arm dips a small tip into DNA and ● Probes can attach to the slide by robotic means
then onto the glass surface to fluorescently ● Microarray spotters are high-precision robots with metal pins
label the sample that dip into DNA solution and taps down on the glass slide
● The probes need to be synthesized prior to ● Glass slides DNA microarrays uses spotting arrays
chip preparation ○ DNA spotting done by robotic arm using multiple pins
● Spotted arrays are the first arrays to be
○ DNA in a microtiter plate
created
● This is the most straightforward to use and ○ DNA usually PC amplified
1. Spotted Array
relatively inexpensive ○ Oligonucleotides can also be spotted plotting
● Allows the user to check the purity and
quantity of the DNA or cDNA to be used ADVANTAGES DISADVANTAGES
● Spotted arrays can be time-consuming as the ● Relatively affordable with a ● Requires intensive labor for
DNA or RNA have to be extracted, converted lower cost synthesizing & purifying DNA
into cDNA (in the case of an RNA), and then ● It requires no specialized solutions before microarray
purified. equipment fabrication
● Prior knowledge of sequence is ● Low density of probe (Slide
○ It is time consuming. not necessary DNA) as compared to
● Good choice for new gene oligonucleotides array
● These arrays use light and photolithography
discovery ● Cross hybridization may
to create probes on chips
● Necessary clone sets are occur due to common
● These probes are oligonucleotides that
available for numerous sequences from same gene
match down sequences of targeted DNA
organisms (humans, mice, rats) family
● These arrays are faster than spotted arrays
2. In-Situ Array and do not need cDNA handling; it only
requires the target sequence to be known 2. DNA Chip (Oligonucleotide) Microarray
● In-situ arrays have higher sensitivity
● In-situ arrays are costly and requires
specialized machinery to synthesize many
arrays themselves
● These arrays are fiber optics
● DNA is assembled on small polystyrene
beads
● Some versions contain wells already
3. Self-Assembled
pre-made in a glass to hold the beads
Array
● The beads are encoded with different
combinations of fluorophore so that the ● The process is done by pre-fabricated or synthesizing single
randomly assembled oligos are identified by stranded oligo
their specific combinations ● Since oligos are usually short, the density of these arrays is
much higher
● Traditionally called, 'Gene Chips' by commercial suppliers, such
as Affymetrix, Agilent
● More advanced technique where oligonucleotides are
synthesized on the chip

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 will see that the gene BRCA1 is directly proportional to
the intensity of the cancer of the patient.
ADVANTAGES DISADVANTAGES ● So, on the slide shown before, we will know because
there is a specific sequence on the slide for the BRCA1
● Fast ● In-situ oligonucleotide array formats tend to gene if it is present. So, all 20,500 spots and sequences
● Specific have expensive specialized equipment to with the help of gene expression strategies, we can
● Reproducible carry out hybridization, staining of label, detect which genes is activated in which tumor. So,
washing, and quantitation process. different abnormalities in our body if we have breast
● Short sequences (oligonucleotides 10-15 cancer or if we have lymphoma, DNA microarray will be
base pair only) used on the array have the one to tell us which cancer we have because different
decreased sensitivity/binding compared caner have different sequence.
with glass cDNA microarrays.
6 Analyzation of Data
DNA MICROARRAY PROCEDURE
1 Collection of Samples
● This can be a variety of organisms
● Samples which can be collected:
a. Peripheral Blood
b. Aspirate Fluid
c. Swab Sample
d. Lavages
● Any cells that has DNA can act as a sample
2 Isolation of 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
● Green - the healthy sample is more hybridized than the
from the rRNA and tRNA. The mRNA has a poly-A tail,
deceased sample
so we can use a column containing beads with poly-T
● Red - the diseased/cancerous sample is more
tails to bind with the mRNA
hybridized than the non-diseased sample
● Rinse with buffer to release the mRNA from the beads.
● Yellow - both samples are hybridized equally to the
The buffer disrupts the pH, disrupting hybrid bonds
target DNA
3 Creation of Labeled DNA ● Black - areas where neither sample are hybridized to
● Add a labelling mix to the RNA. The labelling mix the target DNA.
contains poly-T primers, reverse transcriptase (to make ● This is an example of the printout of a DNA microarray
cDNA), and fluoroscently dyed nucleotides scanner. By comparing the differences in gene
● Add Cyanine-3 (fluoresces green) to the healthy cells expression between 2 samples, we can understand more
● Add Cyanine-5 (fluoresces red) to the cancerous cells about the genomics of a certain disease – like how miss
4 Hybridization mentioned the breast cancer or the BRCA1 gene before.
● Apply the cDNA (complementary DNA) to the microarray
plate DNA MICROARRAY PROCEDURE RECAP
● Apply both samples to the same plate
● The ssDNA (Single Stranded DNA) will bind to the cDNA
already present on the microarray plate
5 Microarray Scanner

● Step where it scans or reads the things contained in the

probes.
● The scanner consist of the following:
○ Laser - causes the hybrid bonds to fluoresce
○ Camera - records the images produced when the
laser scans the plate
1 Source of Sample
○ Computer - allows us to immediately view our
● It must be from any 2 sources of sample.
results and it also stores data
1. Control Sample
■ The scanner scans the green images
2. Experimental Sample
first, then the red images. After, it will
● The normal cell is compared to a malignant or
merge the images and that is now your
preneoplastic cell.
yellow image.
■ The order of sequence is green > red > 2 Extract Messenger RNA
yellow. ● This is the RNA of Interest for DNA Microarray.
● For example, we have the BRCA1 gene or the gene 3 Creation of Labelled DNA by the Addition of two nucleotide
present when you have breast cancer. So if we are trying dyes
to see if the patient has breast cancer, and we will have ● Cyanine 3 - Fluoresces Green
the same results after many runs sa DNA microarray. We ● Cyanine 5 - Fluoresces Red

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 4 Competitive Hybridization and Immunopathology (SLE, Multiple Sclerosis,
5 High Resolution Laser Scanning Organ Transplantation)
● Detects the fluorescent signals from each of the spots. Used to assess the risk that a breast tumor will
Once the samples are mounted on the DNA Microarray spread to the other part of the body (metastasis). The
slide by Microarray spotting, competitive hybridization MammaPrint is based on the well-known 70-gene
MAMMAPRINT
occurs and it gets inside the scanner. breast cancer signature. In February 2007, the FDA
6 Image Analysis cleared the MammaPrint test for use in the United
● There will be a printout/reading and data analysis will States.
follow
OTHER BENEFITS OF MICROARRAY
7 Data Analysis
● Different gene abnormalities in the body have a different ● New Gene Discovery
sequence in the area of DNA Microarray. There is a ● Gene expression profiling
specific sequence for each cancer gene. ● Cancer Diagnosis and Classification
8 Data Interpretation ● Drug Discovery and Toxicology
9 It can then be diagnosed as to what type of cancer he/she has. ● DNA Resequencing
The fluorescent’s intesity of each spot is proportional to the ● Agricultural/Forensic Science
level of expression. For each sample, the expression level of
thousands of genes is obtained and by using the bio informatic DRAWBACKS OF MICROARRAY
tools, the relative levels of gene expression in different samples ● Lack of standardization
can be compared. That is why we need to extract cells from two
● Inadequate computer-based tools
samples. It is either a normal cell or a
cancer/malignant/preneoplastic cell. ● Statistical problem
● TIme required to perform microarrays even at the most automated
SUMMARY INTERPRETATION/ANALYSIS OF RESULTS OF format may still be 3 to 4 days
DNA MICROARRAY ● Variability of outcome
● Effects of probe length
● The ratio of the red and green fluorescent’s intensities for
● Microarray technology needs many longer prospective correlative
each pot is indicative of the relative abundance of the
studies to support the used of microarray-based biomarker panel
corresponding DNA probe in the two nuclei acid target samples.
in routine diagnostic pathology.
○ If the M gene ≤ 0, the gene is overexpressed in
green-labelled samples compared to the red labelled
samples.
○ If the M gene = 0, the gene is equally expressed in both
control and experimental samples.
○ If the M gene＞0, the gene is overexpressed in
red-labelled sample compared to the green labelled
sample which means that the gene is activated in the
tumor cells.
● If it gives off red color or it has more red output,
○ It means that the gene is activated in the tumor cells.
● Whereas if the circle colors black, the gene is not expressed in
both control and experimental samples.

APPLICATIONS OF DNA MICROARRAY

How DNA Microarray is used in every field of Science
Molecular characterization of tumors on a genomic
scale is a more reliable diagnosis and effective
treatment of cancer.
● In the context of cancer, gene expression
profiling has been used to more accurately
CANCER
classify tumors.
RESEARCH
● Each cancer has a specific sequence. Once
results from DNA Microarray are obtained, data
can be analyzed and cancer will then be
classified
● Different cancers have different sequences.
Study of the host genomic responses to bacterial
and viral infections.
● Many viruses/bacteria change their genomic
interaction.
IMMUNOLOGY Ex. HIV - they change their genomic
sequence after being exposed to a certain
chemotherapy agent.
● The ability of the bacteria & viruses to
change their genomic interaction
This technology has been applied to biomarker
NON-TUMOR
discovery in clinical medicine such as neuropathology
PATHOLOGY
(Alzheimer, Parkinsonism, Epilepsy, Schizophrenia)

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