DNA MICROARRAY

TECHNIQUES

Babak Nami. M
 What is DNA Microarray?
• A  DNA microarray (also commonly known as gene chip,
DNA chip, or biochip) is a collection of microscopic DNA
spots attached to a solid surface.
• Scientists use DNA microarrays to measure the
expression levels of large numbers of genes
simultaneously or to genotype multiple regions of a
genome.
 History
• Development of Photolighographic Printing (1991)
• Development of Southern blotting (1992)
• Genomic Libraries (1994)
• Design of first Microarray with special probes (1996)
• Development of DNA Microarray technique (1997)
• Applying DNA Microarray for Cancer study (2000)
• Applications of DNA Microarray in Medicine (2003)
• Development of specific Microarray for Genetics study (2004)
• I am Representing of the technique for Zeynep betül Bulut at
the Selçuk University (2011)
How is microarray manufactured?
• Affymetrix GeneChip
• silicon chip
• oligonucleiotide probes lithographically synthesized
on the array
• cRNA is used instead of cDNA
How does microarray work?
 What is principle?
1. Conjuncture of complementary strand of
target genetic material as Probes on the chips.
2. Preparation unknown DNA/cDNA/cRNA simple
and labeled with a fluorescent dye.
3. Vicinity of Chip and DNA/cDNA.
4. Hybridization of DNA/cDNA with
complementary strain (probe) and remain on
the chip.
5. Capture of emitted light by target DNA/cDNA
by detection device
6. Analysis of lights intensity using a computer
software like SAS, plus-S, STATA and R
Common types of DNA Microarray
• DNA Complementary Spotted

• Oligonucleotide Array

• Microarray CGH
 Applications of DNA Microarray
• Discovery of new genes
• Diagnosis of cancers and genetic
abnormalities
• Discovery of new drugs
• Genetic Screening
• Discovery of genes function
• Genes expression rates
• Detections of viruses
• Detections of pathogen bacteria
• Toxicology
• Classification of cancers like Leukemia
Comparative Genomic Hybridization (CGH)
• preface
• Many human genetic disorders result from unbalanced
chromosomal abnormalities, in which there is net gain or loss of
genetic material. Traditionally, cytologists have detected such
abnormalities by generating a karyotype of a person's
chromosomes and analyzing the banding patterns therein.
• Indeed, since its development in the 1970s, cytogenetic analysis
of banding patterns has been the primary tool for the clinical
assessment of patients with a variety of congenital anomalies.
Under ideal conditions, aberrations as small as approximately 5
megabases (Mb) can be detected with banding analysis; such
chromosome rearrangements are termed "microscopic."
• During the 1990s Thomas Cremer and Peter Lichter
realized the concept of comparative hybridization to
metaphase chromosomes and to a matrix DNA spots
representing specific genomic sites.
• CGH or Chromosomal Microarray Analysis (CMA) is a
molecular-cytogenetic method for the analysis of copy
number changes (gains/losses) in the DNA content of a
given subject’s DNA and often in tumor cells.
• CGH will detect only unbalanced chromosomal changes,
but array CGH can do both balanced and unbalaned.
 Principle
• DNA from subject tissue and from normal control tissue
(reference) are each labeled with different tags for later analysis
by fluorescence.
• After mixing subject and reference DNA along with unlabeled
human cot-1 DNA (placental DNA that is enriched for repetitive
DNA sequences such as the Alu and Kpn family) to suppress
repetitive DNA sequences, the mix is hybridized to normal
metaphase chromosomes or, for array- or matrix-CGH, to a slide
containing hundreds or thousands of defined DNA probes.
• Using epifluorescence microscopy and quantitative image
analysis, regional differences in the fluorescence ratio of
gains/losses vs. control DNA can be detected and used for
identifying abnormal regions in the genome.
array-CGH of a glioblastoma
 Human Cot-1 DNA®
1. COT-DNA (the correct word is as C0t -DNA, The C0t is a value that is the
product of C0 (the initial concentration of DNA), t (time in seconds) and a
constant that depends on the concentration of cations in the buffer.
Repetitive DNA will renature at low C0t values, while complex and unique
DNA sequences will renature at high C0t values).
2. (cot1/2); The product of Co (the original concentration of denatured DNA)
and t (time in seconds), giving a useful index of DNA renaturation
Human Cot-1 DNA®is placental DNA that is predominantly 50 to 300 bp in size
and enriched for repetitive DNA sequences such as the Alu and Kpn family
members.
Human Cot-1 DNA® is commonly used to block non-specific hybridization in
microarray screening.
It can also be used to suppress repetitive DNA sequences for the direct
mapping of human DNA or mapping genomic clones to panels of somatic-cell
hybrids for chromosome localization by Southern blotting.
 Advantages of aCGH Technology
• CGH does not require Chromosomes. DNA isolation
is enough (Speicher et al., 1993).
• CGH does not require cells that are undergoing
division (Speicher et al., 1993).
• Using a conventional CGH, copy number changes at
a level of 5 – 10 kilobases of DNA sequences can be
detected. Today HR-CGH array accurate to detect
SV at resolution of 200 bp. (Oligonucleotide Array
contain the oligos, offer a resolution typically of
20-80 base pairs.)
 Advantages of aCGH Technology
• All 46 chromosome can be examined in a
single test.
• More sensetive and accurate than
conventional karyotyping.
• Simultaneously detect aneuploidies,
deletions, duplications, and/or amplification.
• One CGH = thousands of FISH experiments.
• Attendant saving in labor and expense.
 Advantages of aCGH Technology
• A CGH has a 10% - 20% detection rate of
chromosomal abnormalities in children with
mental retardation. (only 3% - 5% of these
abnormalities would be detectable by other
means).
For example: In a study of 8,789 cases
analyzed by aCGH, 1,049 (11.9%) had a clinically
relevant chromosomal abnormality (Shaffer et
al, 2007).
 Importance of CGH
• Any Cancers
• chromosomal anomalies (Microscopic and
submicroscopic)
• More than 70 congenital anomalies caused by
copy number variants (CNV)
• Prenatal diagnosis such as in Preimplantation
genetic diagnosis and spontaneous
miscarriages.
 Prenatal diagnosis
• In CGH, the nucleus of the embryo is labeled with a
fluorescent dye and a control cell is labeled using
another color (i.e., red or green). 
• The embryo and the control cell are then cohybridized
onto a control metaphase spread, and the ratio
between the two colors is compared. 
• If the chromosomal analysis shows an excess of red, the
embryo nucleus contains an extra chromosome. 
• Currently, this technique takes 72 hours, and embryo
cryopreservation is necessary to provide the time
necessary to undertake the diagnosis. 
 congenital anomalies
• More than 70 disorders are known to be associated
with birth defects or developmental problems that
are caused by deletion or duplication of genomic
material, which are called "copy number variants,"
or CNVs.
• Comparative genomic hybridization is intended to
increase the chromosomal resolution for detection
of CNVs, and as a result, to increase the diagnostic
yield and the genomic detail beyond that of
conventional methods
 subtelomeric rearrangments
• In recent years, aCGH has been particulary useful in
the study of subtelomeric rearrangments
pericentromeric
Note: aCGH data can be verified using FISH analysis.
For Instance:
Ballif and others (2007) recently characterized 169 cases with
subtelomeric abnormalities identified by aCGH.
Although the coverage was sufficient to define the breakpoints
in over half (56%) of the subtelomeric abnormalities, 44% of
the abnormalities extended outside the coverage, suggesting
that many such abnormalities are greater than 5 Mb in size.
 aCGH analysis and FISH combined can identify small subtelomeric
regions associated with clinical phenotypes.

a) aCGH analysis of the genome of "Patient 5" revealed a deletion

in the subtelomeric region of chromosome 17. This deletion was
confirmed using FISH in the patient (b) Chromosome 17 from the
mother and father of the patient is shown in (c) and (d).
 Pericentromeric rearrangments
• Because of the high levels of repetitive
sequences present in the pericentromeric
regions and the variability in presentation
associated with traditional G-banding,
rearrangements in these regions are
inherently difficult to assess by chromosome
analysis
 For instance:
One study of 8,789 individuals with mental retardation
and/or birth defects using aCGH (Shaffer et al., 2007):
94 individuals were found to have a microdeletion in a
pericentromeric region, and 42 individuals were found to
have reciprocal duplications in these regions.
In addition, 22 individuals had novel deletions, while 11
individuals had novel duplications of other pericentromeric
regions that were found in two or more patients.
Among these individuals were four with recurrent de novo
interstitial deletion in band p11.2p12.2 on the short arm of
chromosome 16.
Microdeletion Syndromes caused by NAHR
• Many such microdeletion syndromes are caused by nonallelic
homologous recombination (NAHR) mediated by flanking
segmental duplications (Shaffer et al., 2001)
• The duplications involving segments smaller than 1.5 Mb may
be routinely missed even by FISH of interphase nuclei (Shaffer
et al., 1997)
• However, recent large-population studies of individuals tested
by aCGH have shown that the frequency of reciprocal
duplications is higher than detected in previous studies that
used other cytogenetic technologies (Shaffer et al., 2007; Lu
et al., 2007).