UNIVERSITI MALAYSIA SABAH

FACULTY OF SCIENCE AND HUMAN RESOURCES

HGO7 BIOTECHNOLOGY

SEMESTER 2 (2018/2019)

INSTRUMENTATION AND APPLICATION

“DNA Microarray”

Name: Shalini Muthu Chalvan

Matric number: BS17110173

Lecturer’s name: Dr Suraya Abd

Brief history of DNA Microarray

In 1995, the first study that used the word 'microarray' was published which explained how
the expression of many genes could be monitored in parallel through the use of this new
technology. The sample array was constructed through high-speed robotic printing of cDNA
on glass. The small size of spots on the array and high density of the arrays produced
hybridization volumes of two microliters, which was the volume that enabled the detection of
rare transcripts within the probe samples. The microarray was a technical advancement that
meant a broader examination of gene expression could be accomplished. In 1997, the
researchers from Stanford University published the first whole-genome microarray study of
gene expression by placing the whole yeast genome on a microarray.

The technology was not commercialized and the Stanford University laboratory published the
instructions for producing the robotics necessary for a microarray. While today there are
commercial microarray options with greater sensitivity, the simple production of microarrays
has allowed for easily customizable experiments within laboratories.

Introduction to DNA microarray

Molecular Biology research evolves through the development of the technologies used for
carrying them out. It is not possible to research on a large number of genes using traditional
methods. DNA Microarray is one such technology which enables the researchers to investigate
and address issues which were once thought to be non-traceable. One can analyse the
expression of many genes in a single reaction quickly and in an efficient manner. DNA
Microarray technology has empowered the scientific community to understand the
fundamental aspects underlining the growth and development of life as well as to explore the
genetic causes of anomalies occurring in the functioning of the human body.

A typical microarray experiment involves the hybridization of an mRNA molecule to the DNA
template from which it is originated. Many DNA samples are used to construct an array. The
amount of mRNA bound to each site on the array indicates the expression level of the various
genes. This number may run in thousands. All the data is collected and a profile is generated
for gene expression in the cell.
What is Microarray?

Microarray technology has become one of the indispensable tools that many biologists use to
monitor genome wide expression levels of genes in a given organism. A microarray is typically
a glass slide on to which DNA molecules are fixed in an orderly manner at specific locations
called spots (or features). A microarray may contain thousands of spots and each spot may
contain a few million copies of identical DNA molecules that uniquely correspond to a gene
(Figure A). The DNA in a spot may either be genomic DNA or short stretch of oligo-nucleotide
strands that correspond to a gene. The spots are printed on to the glass slide by a robot or
are synthesised by the process of photolithography. Microarrays may be used to measure
gene expression in many ways, but one of the most popular applications is to compare
expression of a set of genes from a cell maintained in a particular condition (condition A) to
the same set of genes from a reference cell maintained under normal conditions (condition
B).
(Figure B) gives a general picture of the experimental steps involved.

1. RNA is extracted from the cells.

2. RNA molecules in the extract are reverse transcribed into cDNA by using an enzyme
reverse transcriptase and nucleotides labelled with different fluorescent dyes.
3. As in the above figure, cDNA from cells grown in condition A may be labelled with a
red dye and from cells grown in condition B with a green dye
4. Once the samples have been differentially labelled, they are allowed to hybridize onto
the same glass slide.
5. Any cDNA sequence in the sample will hybridize to specific spots on the glass slide
containing its complementary sequence.
6. The amount of cDNA bound to a spot will be directly proportional to the initial number
of RNA molecules present for that gene in both samples.

Microarray principle

A mRNA is an intermediary molecule which carries the genetic information from the cell
nucleus to the cytoplasm for protein synthesis. Whenever some genes are expressed or are
in their active state, many copies of mRNA corresponding to the particular genes are produced
by a process called transcription. These mRNAs synthesize the corresponding protein by
translation. So, indirectly by assessing the various mRNAs, we can assess the genetic
information or the gene expression. This helps in the understanding of various processes
behind every altered genetic expression. Thus, mRNA acts as a surrogate marker. Since mRNA
is degraded easily, it is necessary to convert it into a more stable cDNA form. Labelling of
cDNA is done by fluorochrome dyes Cy3 (green) and Cy5 (red). The principle behind
microarrays is that complementary sequences will bind to each other.

The unknown DNA molecules are cut into fragments by restriction endonucleases; fluorescent
markers are attached to these DNA fragments. These are then allowed to react with probes
of the DNA chip. Then the target DNA fragments along with complementary sequences bind
to the DNA probes. The remaining DNA fragments are washed away. The target DNA pieces
can be identified by their fluorescence emission by passing a laser beam. A computer is used
to record the pattern of fluorescence emission and DNA identification. This technique of
employing DNA chips is very rapid, besides being sensitive and specific for the identification
of several DNA fragments simultaneously.
Instrumentation of Microarray
Applications of Microarray

1. Gene expression analysis

The predominate application of DNA microarrays has been to measure gene expression levels.
In this application, RNA is extracted from the cells of interest and either, labelled directly,
converted to a labelled cDNA or converted to a T7 RNA promoter tailed cDNA which is further
converted to cRNA through the Eberwine amplification process (Van Gelder et al., 1990). A
wide variety of methods have been developed for labelling of the cDNA or cRNA including:
incorporation of fluorescently labelled nucleotides during the synthesis, incorporation of biotin
labelled nucleotide which is subsequently stained fluorescently labelled streptavidin,
incorporation of a modified reactive nucleotide to which a fluorescent tag is added later, and
a variety of signal amplification methods (an early review of different labelling methods is
provided in (Richter et al., 2002)). The two most frequently used methods are the
incorporation of fluorescently labelled nucleotides in the cRNA or cDNA synthesis step or the
incorporation of a biotin labelled nucleotide in the cRNA synthesis step

The labelled cRNA or cDNA are then hybridized to the microarray, the array is washed and
the signal is detected by measuring fluorescence at each spot. In the case of biotin labelled
samples, the array is stained post-hybridization with fluorescently labelled streptavidin. Laser
induced fluorescence is typically measured with a scanning confocal microscope.
2. Cancer

Tumour formation involves simultaneous changes in hundreds of cells and variations in genes.
Microarray can be a boon to researchers as it provides a platform for simultaneous testing of
a large set of genetic samples. It helps especially in the identification of single-nucleotide
polymorphisms (SNPs) and mutations, classification of tumours, identification of target genes
of tumour suppressors, identification of cancer biomarkers, identification of genes associated
with chemoresistance, and drug discovery. For example, we can compare the different
patterns of gene expression levels between a group of cancer patients and a group of normal
patients and identify the gene associated with that particular cancer.

3. Antibiotic treatment

As there is icrease in the number of resistant bacteria and superadded infections has led to
failure of antibiotics. Virulence of the bacterial strains too affects the outcome of the disease
process. In oral cavity where anaerobic bacteria might be the infective agent, they often are
not easily culturable, especially organisms such as actinomyces. DNA microarray analysis helps
as the bacterial genomic DNA often outlasts the viability of the bacteria and a diagnosis can
be made using a small amount of DNA, as opposed to the large numbers of bacteria needed
for culture. In future, an abscess specimen might be sent not for culture and sensitivity testing,
but rather for DNA microarray analysis.
Limitations of DNA Microarray

At their core, microarrays are simply devices to simultaneously measure the relative
concentrations of many different DNA or RNA sequences. As they have been incredibly useful
in a wide variety of applications, they have a number of limitations.

1. The arrays provide an indirect measure of relative concentration. That is the signal
measured at a given position on a microarray is typically assumed to be proportional to
the concentration of a presumed single species in solution that can hybridize to that
location. However, due to the kinetics of hybridization, the signal level at a given location
on the array is not linearly proportional to concentration of the species hybridizing to the
array. At high concentrations the array will become saturated and at low concentrations,
equilibrium favours no binding. Hence, the signal is linear only over a limited range of
concentrations in solution.
2. Considering the complex mammalian genomes, it is often difficult to design arrays in which
multiple related DNA/RNA sequences will not bind to the same probe on the array. A
sequence on an array that was designed to detect “gene A”, may also detect “genes B, C
and D” if those genes have significant sequence homology to gene A. This can particularly
problematic for gene families and for genes with multiple splice variants.
References

Bussemaker, H. J., Li, H., and Siggia, E. D. (2001). Regulatory element detection using
correlation with expression. Nat. Genet. 27, 167-171.

M. Madan Babu. (n.d). An Introduction to Microarray Data Analysis. Retrieved from

https://www.mrc-lmb.cam.ac.uk/genomes/madanm/microarray/chapter-final.pdf

Roger Bumgarner. DNA microarrays: Types, Applications and their future. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4011503/

Shelley Farrar Stoakes. (2019). History of Microarrays. Retrieved from https://www.news-

medical.net/life-sciences/History-of-Microarrays.aspx