LECTURE \ VELEZ COLLEGE \ MT2

LECTURE 10: DNA MICROARRAY

OUTLINE
I.DNA Microarray
A. Differentiate Genetics VS Genomics
B. Define what microarray is
C. Identify the different types of DNA Microarrays
D. Discuss the procedures of DNA Microarray
E. Discuss the applications of DNA Microarray

GENETICS
● “Study of Heredity”
● It focuses primarily on the likelihood of developing cancer
● Genetics tests finds mutations, not disease

GENOMICS ● Each human cell contains 46 chromosomes and each

chromosome has 20,500 genes
● “Study of genes”
● How they interact and expressed as a whole
● Genetics and genomics are profiling tools that focuses on STRUCTURAL GENES VS REGULATORY GENES
cancer itself and can help determine: ● Please take note of the examples.
1. How aggressive the cancer is (prognosis)
2. What is the likely benefit from the treatment Structural Genes Regulatory Genes
(prediction) A gene that codes for any A gene involved in
RNA or protein product controlling the expression of
Human Genomics Project other than a regulatory one or more other genes
● It is an international scientific research project factor
● Determining the sequence of human DNA Encode for proteins or RNA Encode for one or more
● Identifying and mapping all the human genes transcription factors or
● Key findings: There are approximately 20,500 genes in a regulatory RNA
human being Encode for mRNAs, rRNAs, Encode for regulatory RNA
and tRNAs such as miRNAs and
GENES siRNAs
● They are a part of a DNA Gene products have either Regulate the expression of
structural or functional structural genes
● They are the basic physical and functional unit of heredity
importance
● Genes are made up of DNA. Some genes act as
They decide if ‘ah pwede na
instructions to make molecule called proteins
ni iactivate ang actin na
● The nucleotide sequence is (A-T,C-G) on the DNA
gene para maka produce na
sha ug protein.’

Regulatory genes ensure

Examples: Laz Z, lac Y, and
lac A genes of the lac
operon, actin gene, etc.
that appropriate genes are
expressed at a proper time.
Examples: Lac I gene, CAP
gene, etc.

WHOLE DNA OR GENES

A whole DNA or gene is divided into two parts:

Intron
● The first part is the Intron wherein it is a portion of a gene
that does not code for amino acids or proteins.

Exon
● Next we have the exon. It is a portion of a gene that will form
a part of the final mature RNA produced by that gene after
introns have been removed by RNA splicing.
o So in short, ang intron kay intermission ra ni sha.
Murag they are there lang para nay time mo kanang
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maka form ug final mature RNA ang whole DNA. So
the Introns would later be removed by RNA splicing.
o So here mao ni sha ang example. So exon and then
intron and then exon. So in between two exons is one
intron. Ang intron is a portion of a gene that does not
code for any amino acids or proteins. So kini sha ma
remove ra ni siya later on by RNA splicing.
GENE EXPRESSION
● Gene Expression occurs only in a particular type of cell or
tissue where only a subset of an organism’s DNA will be
expressed as mRNAs at any given time.
o So ang Central Dogma of Life is from the DNA to the
RNA to protein. So ang DNA niya ma activated sha by
the RNA. Ara pa sha mahimo ug protein. So the central
dogma of life is very interesting to know the expression
of the gene.
● The gene expression is focused on the Central Dogma of
Life where the 20,500 genes are activated simultaneously
and these genes are only activated when they are already
needed.
o For example, after eating food, our body needs insulin
so the genes in our pancreas get activated and then
they send mRNA to activate the code of sequence for
protein synthesis. So from DNA, mu send siya
message to mRNA, after ma activate maka produce na
ug insulin atoang pancreas.
● In gene expression, PROTEINS DO MOST OF THE
WORK.
o Proteins are either dynamically created or destroyed
o mRNA blueprint are also dynamically created or
destroyed
o Different mRNAs expressed at different times/ places
(So different sad siya na proteins ang ilahang ma
produce, so that is why it is important to know the
mRNA expression levels)
o Important to know the mRNA expression levels
● The unique pattern of gene expression for a given cell or
tissue is referred to as molecular signature
● Why is gene expression important in any
malignancy/disease? The use of gene expression
profiling and development of gene biomarkers/signatures
for cancer allows for the diagnosis, progression, and
aggressiveness analyses, prognosis, and prediction of
therapeutic treatment.
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So here, ato ma relate ang gene expression and cancer where
a normal cell is compared to a cancer cell. In a normal cell, only
gene A and gene C are being activated while for the cancer cell,
only gene B and gene C are activated. So kung ma activate ang
gene B, meaning, naay cancer ang patient. After sa gene
expression, once ma activate ang gene B, that is an indication
of cancer.
GENE EXPRESSION ASSAYS
The main types of gene expression assays are:
● Serial Analysis of Gene Expression (SAGE)
● Short Oligonucleotide Arrays (Affymetrix)
● Long Oligonucleotide Arrays (Agilent Inkjet)
● Fiber Optic Arrays (Ilumina)
● cDNA Arrays (Brown/Bowstein)
DNA MICROARRAY HISTORICAL BACKGROUND
● Mark Schena - Father of Microarray Technology
● Steve Fooder - developed a scanner for reading the output
● Patrick Brown and Mark Schena - Quantitative monitoring
of Gene Expression 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
DNA MICROARRAY
● Commonly known as “DNA Chip” or biochip
● Is composed of a collection of microscopic DNA steps
attached to a solid surface
● Is a technology that can sequence thousands of genes at a
time in order to quantify the expression of the said genes
● Microarray technology uses a microarray chip which
contains probes at different locations on its surface that can
bond to target DNA
● The bonding of probes and targets is referred to as
“HYBRIDIZATION”
● Microarrays can be used to study gene expressions in
different conditions or in different cells
● Target DNA - this is the DNA of a sample of interest which
is compared to a reference DNA or ‘control DNA’
● The differential binding and expression of fluorescent
probes on the chip allows the user to determine the relative
expression of target genes
DNA MICROARRAY PROCEDURES
● Relies on the hybridization properties of nucleic acids to
monitor DNA or RNA abundance on a genomic scale in
different types of cells.
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Principle of DNA MICROARRAY
● The core principle behind microarrays is the hybridization
between 2 DNA strands where fluorescent labeled cDNA or
nucleotide (target) binds, to a DNA on the slide (probe), will
then generate a signal that depends on the strength of the
hybridization determined by the number of paired bases.
● The gene that is placed in the glass slide is called a probe.
● Microarrays in general are a snapshot that captures the
activity patterns of thousands of genes at once and
microarrays also allow simultaneous measurement of the
level of the transcription of every gene in a genome.
● This is what we consider as a gene expression.
The ARRAY/SLIDE
● 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 spots per square inch
● Each spot holds millions of copies of a DNA sequence from
one gene
● An array is composed of
multiple tiny dot
● Each dot contains one
complimentary copy or one
single probe of DNA
● Mao ni siya e use for
conventional slide method.
3 MAIN TYPES OF DNA MICROARRAY
1. SPOTTED ARRAY
● These arrays use a poly-lysine coated microscope
slide
● A robotic arm dips a small tip into DNA and then on to
the glass surface to fluorescently label the sample
● The probes need to be synthesized prior to chip
preparation
● Spotted arrays are the first arrays to be created since
this is the most straightforward to use and relatively
inexpensive
● Allows the user to check the purity and quantity of the
DNA or cDNA to be used
● Spotted arrays can be time-consuming as the DNA or
RNA have to be extracted, converted into cDNA (in the
case of an RNA), and then purified.
2. IN-SITU ARRAY
● These arrays use light and photolithography to create
probes on chips
● These probes are oligonucleotides that match down
sequences of targeted DNA
● These arrays are faster than spotted arrays and do not
need cDNA handling; it only requires the target
sequence to be known
● In-situ arrays have higher sensitivity
● In-situ arrays are costly and requires specialized
machinery to synthesize many arrays themselves
3. SELF-ASSEMBLED ARRAY
● These arrays are fiber optics
● DNA is assembled on small polystyrene beads
● Some versions contain wells already pre-made in a
glass to hold the beads
● The beads are encoded with different combinations of
fluorophore so that the randomly assembled oligos are
identified by their specific combinations
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2 COMMON METHODS OF DNA MICROARRAY
1. GLASS SLIDE cDNA MICROARRAY
● Conventional method
● First type of DNA Microarray technology developed
● Invented by Patrick Brown & his colleagues at Stanford
University
● Uses a robotic device which deposits (spots) a
nanoliter sample of DNA onto a coated microscopic
glass slide (50-150 micrometer in diameter)
● The slide is usually made commercially, and is made
of glass/silicon/nylon
● Each slide contains thousands of spots, and each spot
contains a probe for a different gene
● Probes can attach to the slide by robotic means
● Microarray spotters are high-precision robots with
metal pins that dip into DNA solution and taps down on
the glass slide
● Use spotting/spotted arrays
○ DNA spotting done by robotic arm using multiple
pins
○ DNA in a microtiter plate
○ DNA usually PCR amplified
○ Oligonucleotides can also be spotted
Advantages:
● Relatively affordable with a lower cost
● Requires no specialized equipment
● Prior knowledge of sequence is not necessary
● Good choice for new gene discovery
● Necessary clone sets are available for numerous
organisms (humans, mice, rats)
Disadvantages:
● Requires intensive labor for synthesis and purification of
DNA solutions before microarray fabrication
● Low density of probe (slide DNA) as compared to
oligonucleotides array
● Cross hybridization may occur due to common sequences
from same gene family
2. DNA CHIP (OLIGONUCLEOTIDE) MICROARRAY
● Process is done by pre-fabricated or synthesizing
single stranded oligo
● Since oligos are usually short, the density of these
arrays is much higher
● Traditionally called ‘gene chips’ by commercial
suppliers such as Affymetrix and Agilent
● More advanced technique where oligonucleotides are
synthesized on the chip
Advantages: Fast, Specific, Reproducible
Disadvantages:
● In-situ oligonucleotide array formats tend to have expensive
specialized equipment to carry out hybridization, staining of
label, washing, and quantitation process
● Short sequences (oligonucleotides contain 10-15 base
pairs only) used on the array have decreased
sensitivity/binding compared to glass cDNA microarrays
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DNA MICROARRAY PROCEDURES ● Different abnormalities in the body if you have breast
cancer or lymphoma, DNA Microarray will be the one to tell
1. COLLECTION OF SAMPLES us which cancer we have because different cancers have
different sequences
● Can be a variety of organisms
● Any cell that has DNA can act as a sample
● Samples which can be collected:
○ Peripheral Blood
○ Aspirate Fluid
○ Swab Sample
○ Lavages

2. ISOLATION OF MRNA
● mRNA is the RNA of interest because it contains the code
for protein synthesis Microarray Scanner
● Extract the RNA from the samples. Using either a column
or a solvent such as phenol chloroform 6. ANALYZATION OF DATA
● After isolating the RNA, we need to isolate the mRNA from
the rRNA and tRNA. The mRNA has a poly-A tail, so we
can use a column containing beads with poly-T tails to bind
with the mRNA
o A always binds with T
● Rinse with a buffer to release the mRNA from the beads.
The buffer disrupts the pH, disrupting hybrid bonds
o Cells contain all the RNA (mRNA, rRNA, tRNA) that is
why we need to isolate the mRNA because that is the
RNA of interest because the mRNA contains the code
for protein synthesis
Sample image of the DNA Microarray scanner
3. CREATION OF LABELED DNA
Possible results seen in the print out:
● Add a labeling mix to the RNA. The labeling mix contains ● GREEN - the healthy sample is more hybridized than the
poly-T primers, reverse transcriptase (to make cDNA), and diseased sample
fluorescently dyed nucleotides ● RED - the diseased/ cancerous sample is more hybridized
● Add Cyanine-3 (fluoresces green) to healthy cells than the non-diseased sample
● Add Cyanine-5 (fluoresces red) to the cancerous cells ● YELLOW - both samples are hybridized equally to the
target DNA
4. HYBRIDIZATION ● BLACK - areas where neither sample are hybridized to the
● Apply the cDNA to the microarray plate target DNA
● Apply both samples to the same plate ● By comparing the differences in gene expression between
● The ssDNA will bind to the cDNA already present on the two samples, we can understand more about the genomics
microarray on the plate of a certain disease.
o For example, breast cancer or the BRCA1 gene. It has
5. MICROARRAY SCANNER a specific sequence for the disease para madetect jud
● The scanner consists of the following: siya sa DNA Microarray.
a. Laser - causes the hybrid bonds to fluoresce
b. Camera - records the images produced when the laser
scans the plate
c. Computer - allows us to immediately view our results
and it also stores data
● The scanner scans the green images first, and then the red
images, and then it will merge images mao na siya ang
yellow
● Order of Sequence: Green > Red > Yellow
● Example: Gene BRCA1 or the gene present when you have
breast cancer. If you are trying to see if the patient as breast
cancer, and you have the same result after many runs of
the DNA Microarray, we will see that the gene BRCA1 is
directly proportional to the intensity of the cancer of the
patient
o Ganina sa slide, we will know that there is a specific
sequence for the BRCA1 gene. If present, there is a
specific sequence on the slide.
● All 20,500 spots and sequences with the help of gene
expression strategies, we can detect which gene is
activated in which tumor.

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SUMMARY OF THE INTERPRETATIONS/ ANALYSIS

OF RESULTS IN DNA MICROARRAY
● The ratio of red and green fluorescence intensities for each
spot is indicative of the relative abundance of the
corresponding DNA probes in the nucleic acid target
samples.
● If the M gene is ≤ 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
samples.
● If the M gene is ≥ 0, the gene is overexpressed in red
labelled samples compared to the green labelled samples,
which means that the gene is activated in the tumor cells.
o If mas daghan galig red na output and DNA Microarray,
it means that the gene is activated in the tumor cells.
● If the circles in the DNA Microarray printout are colored
black, it means that the gene is not expressed in both the
control and experimental samples.

APPLICATIONS OF DNA MICROARRAY

Cancer Research
● Molecular characterization of tumors on a genomic scale is
a more reliable diagnosis and effective treatment of cancer.
DNA Microarray ● In the context of cancer, gene expression profiling has been
used to more accurately classify tumors.
Review of the steps: ○ Remember that each tumor/cancer has a specific
● As mentioned, we always have 2 sources of sample: sequence because different cancers have different
o Controlled sample sequences. Once we get the result of the DNA
o Experimental sample Microarray, we can analyze the data and see whether
● The samples are either from a normal cell then we compare a sequence corresponds to a cancer.
it to a malignant cell or a pre-neoplastic cell.
● Once you have your sample, you need to extract the Immunology
messenger RNA (mRNA) because it is the RNA of ● Study of the host genomic responses to bacterial and viral
interest for DNA Microarray. infections.
● Once you have extracted the mRNA, you will then proceed ○ Many viruses and bacteria change their genomic
with the creation of labelled DNA by the addition of 2 interactions. ex. HIV changes its genomic sequence
nucleotide dyes: after being exposed to a certain chemotherapy agent.
o Cyanine 3 (Cy3) - which fluoresces green
o Cyanine 5 (Cy5) - which fluoresces red Non-Tumor Pathology
● Once the nucleotide dyes are added, we proceed with ● This technology has been applied to biomarker discovery in
competitive hybridization. clinical medicine such as:
● After hybridization, high resolution laser scanning detects ○ Neuropathology: Alzheimer, Parkinsonism, Epilepsy,
the fluorescent signals from each of the spots. Schizophrenia
o Once gani mamount na ang sample on the DNA ○ Immunopathology: Systemic Lupus Erythematosus
Microarray nga slide by Microarray spotting, kay (SLE), Multiple Sclerosis, Organ Transplantation
Competitive hybridization then mosulod na siya sa
scanner. MammaPrint
● It is then followed by image analysis. ● Used to assess the risk that a breast tumor will spread to
● Once you have the print out, Data Analysis will then follow. the other part of the body (will metastasize/undergo
● As mentioned, different gene abnormalities in the body metastasis).
have a different sequence. Different sequences for each ● The MammaPrint is based on the well-known 70-gene
different cancer gene. breast cancer signature.
● Once data is analyzed and interpreted, then we can now ● In February 2007, the FDA cleared the MammaPrint test for
diagnose the patient according to what type of cancer use in the United States.
he/she has.
● The fluorescence intensity of each spot is proportional to OTHER BENEFITS OF DNA MICROARRAY
the level of expression.
1. New gene discovery
● For each sample, the expression level of thousands of
2. Gene expression profiling
genes is obtained. And by using the bioinformatic tools, the
3. Cancer diagnosis and classification
relative levels of gene expression in different samples can
4. Drug discovery and toxicology
be compared. That is why duha ka samples atung kuhaan
ug cells: Control (normal) and experimental 5. DNA resequencing
6. Agricultural/forensic science
(cancer/malignant/pre-neoplastic cell)

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DRAWBACKS OF DNA MICROARRAY

1. Lack of standardization
2. Inadequate computer based tools
3. Statistical problems
4. Time required to perform microarrays even at the most
automated format may still be 3 to 4 days
5. Variability of outcome
6. Effects of probe length
○ Different effects on different lengths of the probe
7. Microarray technology needs many longer prospective
correlative studies to support the use of microarray based
biomarker panel in routine diagnostic pathology

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