Course title: Microbial Genetics and Molecular Biology

Course code: MCB 301

Course lecturer: Dr. O.R. Ogunremi

RECENT TECHNIQUES IN MICROBIAL GENETICS: DNA MICROARRAY TECHNIQUE

Already the whole genome of thousands of microorganisms on publicly

accessible databases. The genome of more than 12,000 bacteria, 100s of fungi
and many viruses have been sequenced. Once the whole genome sequence
of different species of microorganisms was completed, it paved the way for
many experiments and researches to identify the regions of DNA which control
specific phenotype. In addition, it became possible to known gene function
through expression measurements of a genome. The most common tool used
to carry out these measurements is DNA microarrays.

DNA microarrays are technologies in which of sequences of nucleic acids are

bound to hundreds of spots on a surface and are used to detect and measure
the relative concentration of nucleic acid sequences in a mixture via
hybridization and subsequent detection of the hybridization events. This
technique makes use of the principle of hybridization between two DNA
strands that have complementary nucleic acid sequences. Complementary
nucleotide base pairs will specifically pair with each other by forming hydrogen
bonds.

By immobilizing different sequences in unique locations on a solid substrate it is

possible to detect and measure relative levels of many target DNA or RNA
molecules in a complex mixture

A typical DNA microarray is a solid supports, usually of glass or silicon, upon

which millions of copies of specific DNA sequences from one gene are
immobilized on hundreds of spots in an organized pre-determined grid fashion
(rows and columns) such that the identity of each DNA sequence is known
through the location on the support. Each spot of DNA copies is called a probe
and it represents a single gene. A probe is a short section of specific gene or
other DNA element which can be 1000s of nucleotide long that is used to
hybridize a mixture of labelled cDNA or cRNA sample (called target) under
high-stringency conditions.

DNA microarrays can also be called by different names, they include; DNA
chips, gene chips, DNA arrays, gene arrays and biochips.

DNA microarrays can analyze the expression of tens of thousands of genes

simultaneously.

Principle of DNA Microarray Technique

The principle of DNA microarray lies in the hybridization between nucleic acid
strands. Complementary nucleic acid sequences are able to specifically pair
by forming hydrogen bonds based on the base pairing rule.

Messenger RNA (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. mRNA acts as a surrogate marker.
Since mRNA is degraded easily, it is necessary to convert it into a more stable
complementary DNA (cDNA) form by a process called reverse transcription.
For this, samples presumed to contain specific mRNA, which has been
converted to corresponding cDNA are labelled using fluorescent compounds
(fluorophore), silver, chemiluminescence compound or radioactive
compound. The most commonly used labels are fluorescent compound.
Examples of fluorescent compound are fluorochrome dyes Cy3 (green) and
Cy5 (red). The samples are brought in contact with different probes located
on the DNA microarray. cDNA in the sample that is complementary to specific
probe on the chip will hybridize under stringent conditions such as
temperature, i.e. they will get paired via hydrogen bonds. The non-specific
bonding sequences while remain unattached and washed out during the
washing step of the process. Fluorescently labelled target sequences that bind
to a probe sequence generate a signal. The intensity of the signal, from a spot,
depends upon the amount of nucleic acid sequence (cDNA) in the target
sample binding to the probes present on that spot.

DNA Microarray procedure

The reaction procedure of DNA microarray takes places in several steps:

Collection of samples

1. The sample may be a cell of the microorganism that we wish to conduct

the study on.

Isolation of mRNA

1. RNA is extracted from the sample using a column or solvent like phenol-
chloroform.
2. From the extracted RNA, mRNA is separated leaving behind rRNA and
tRNA.
3. As mRNA has a poly-A tail, column beads with poly-T-tails are used to
bind mRNA.
4. After the extraction, the column is rinsed with buffer to isolate mRNA from
the beads.

Creation of labelled cDNA

1. To create cDNA (complementary DNA strand), reverse transcription of

the mRNA is done.
2. Both the samples are then incorporated with different fluorescent dyes
for producing fluorescent cDNA strands. This helps in distinguishing the
sample category of the cDNAs.

Hybridization

1. The labelled cDNAs from the samples are placed in the DNA microarray
so that each cDNA gets hybridized to its complementary strand; they
are also thoroughly washed to remove unbounded sequences.
Collection and analysis

1. The collection of data is done by using a microarray scanner.

2. This scanner consists of a laser, a computer, and a camera. The laser
excites fluorescence of the cDNA, generating signals.
3. When the laser scans the array, the camera records the images
produced.
4. Then the computer stores the data and provides the results immediately.
The data thus produced are then analyzed.
5. The difference in the intensity of the colours for each spot determines the
character and concentration of the gene in that particular spot.

DNA microarray

Applications of DNA microarrays

1. Expression profiling: Gene expression, which includes two processes,
namely transcription of data from a DNA template to mRNA, and
translation, involving the construction of proteins on the basis of the
information about the linear sequence of their amino acids encoded in
 the mRNA, lies behind all functions and structures of cells in organisms.
DNA microarray technology is used to measure gene expression by
measuring levels of mRNA in biological samples, for example microbial
cells. RNA molecules in the samples are labeled with fluorescent dyes by
using appropriate techniques and presented to an array of spots, where
they hybridize complementary-DNA (cDNA) fragments corresponding to
known coding DNA sequences. The measurement of RNA levels is based
on the fundamental property of nucleotide sequences, which is binding
(hybridizing) to their complements. If the level of the RNA product
corresponding to the DNA placed at a spot X in the microarray is high in
the sample being analyzed then we should observe a high fluorescence
signal at spot X.
Gene expression profiling has been successfully used in monitoring
cellular process, measuring the response of cells or tissues to therapeutic
agents, classification or detection of disease symptoms.
2. Genotyping: Assessing genome content in different cells or closely
related organisms