There are two ways to study any living
organism. Traditional biology studies the whole living organism and its
interactions within the population. This is called as Top-down approach.
Molecular biology on the other hand, focus mainly on the biomolecules which
makes up any organism. This is called as bottom-up approach. Both the methods
are considered as valid although, recent advances in technology has enabled
scientists to concentrate more on the biomolecules.
Proteins and nucleic acids are
considered as the most important biomolecules of any living organism.
Nucleic acid such as DNA stores the
necessary information required by all living organisms to carry out their
biological activities.
Proteins are known as functional unit
of life. They are responsible for catalyzing most of the cellular reactions.
They also help in maintaining the cellular structure and rigidity.
Other biomolecules like carbohydrates
and lipids are also studied in molecular biology for their interactions with
different proteins and nucleic acids.
Study of these biomolecules has their
own benefits. They provide more predictable results to the researchers studying
any living organism.
Study of whole organism gives a more
unpredictable results as there are thousands of other molecules and external
factors which influence the experimental results.
Molecular biology provides scientists
with a toolkit to study the way life works.
- They may use it to study the effect of single gene or protein on the organism.
- They may also study the effect of mutation in a single gene or protein.
- Molecular biology is also used to study when and why certain genes are “switched on or off” in any organism.
All these molecular studies provide
researchers a deeper knowledge of the way life works.
Central dogma of Molecular biology
Central dogma of molecular biology
explains the flow of information from store house i.e. DNA to the expressional
unit i.e. Proteins. This is normally a two-step process.
According to the central dogma of
molecular biology, the flow of information is always from DNA to RNA to
Proteins.
In the first step, one of the strands
of DNA is used as a template to synthesize RNA. This process of synthesis of
RNA from DNA template is known as “transcription”.
Once the RNA is transcribed from DNA
template, the next step is carried out. In the second step, the newly synthesized
RNA is used as a template to synthesis proteins. This process is of synthesis
of protein from RNA template is called as “translation”.
The process of translation is carried
out in a specialized organelle called as ribosomes.
During the process of cell division,
every cell carries out one more complex procedure. During cell division, cells
make copy of their genetic material. This process is called as “replication”.
The process of replication is also
considered as a part of central dogma of molecular biology.
So, we can summarize the central dogma
of molecular biology as follow:
The flow of
information is always from DNA to RNA to proteins. Every cell replicates its
DNA during cell division. These replicated DNA is then transcribed to produce
RNA which in turn is used as template to synthesize proteins.
Every cell follows the same principle
of central dogma. However, there are exceptions to this principle. One of the
famous examples is the “Retrovirus”.
Retrovirus belongs to a group of
viruses which have RNA as their genetic material. They are known to carry a
special enzyme called “Reverse Transcriptase”. When this virus infects
its host, they synthesize a complimentary DNA by using RNA as a template. This
process is mediated by the enzyme Reverse Transcriptase. This complimentary DNA
(also called as cDNA) is then used as template to synthesize RNA followed by
the synthesis of proteins.
Therefore, the
flow of information in such type of viruses is from RNA to cDNA to RNA to
proteins.
Relationship of molecular biology with other biological sciences
Molecular biology shares relationship
with different biological sciences. There is no hard line defining these
disciplines. Researchers uses different techniques which are native to
molecular biology. However, most of the times, these native techniques have to
be used in combination with other techniques borrowed from various biological
disciplines.
The most related biological fields to
molecular biology are Biochemistry and Genetics.
- Biochemistry: - Biochemistry involves the
study of the chemical processes that occur in living organisms with the
ultimate aim of understanding the nature of life in molecular terms. One of the
simplest examples is the study of proteins and its function in cellular
metabolism.
- Genetics: - Genetics is the study of genes and their effect on the living organisms. Researches are more focused on studying individual genes and their functions. One example of genetical studies which is carried out more commonly is the study of mutant genes with respect to normal genes. Genetics also involves the study of knock-down effect of any genes on the living organisms.
- Molecular Biology: - Molecular biology is focused on the molecular understanding of the processes of replication, transcription and translation of genetic material of a living organism. It also involves the study of different cellular functions.
The relationship between these three
major biological sciences is depicted in the picture above. The discipline of
molecular biology overlaps with that of biochemistry and genetics and in many
respects the aims of these disciplines complement each other.
In the recent years, the use of
computers and different computer programmes (software) have ease time
management for researchers.
Since much of the molecular biology is
quantitative, this field also overlaps with bioinformatics and computational
biology.
Common molecular biology techniques
The following list covers some very
basic techniques used in molecular biology.
Polymerase chain reaction: -
Kary Mullis
invented a new method that made it possible to synthesize large quantities of
DNA fragments without cloning. This process is called PCR amplification. The
PCR technique can be compared to a xerox machine in which many copies can be
made of the same DNA sequence.
The various
requirements for the PCR process are: -
- Primers (Both reverse and forward in excess)
- Template DNA
- Taq. Polymerase enzyme
- 25mM MgCl2
- PCR buffers
- Nucleotides (dNTPs)
The
template DNA can be isolated from the pure culture cell palate by conventional
phenol-chloroform-isoamyl alcohol isolation technique or by using kits.
Specialized kits can be used to isolate DNA directly from food, urine, blood,
feces and soil.
The
nucleotide sequence of at least short DNA segment on each side of gene of
interest should be known. Based on these sequences, the complimentary forward
and reverse primers can be synthesized.
A
DNA polymerase lacking 5’ - 3’ exonucleases activity and which is stable and
functional at high temperature is used. E.g. Taq. Polymerase from Thermus
aquaticus, Vent. Polymerase from Thermococcus literolis, Pfv.
Polymerase from Pyrococcus furiosus.
MgCl2
act as a cofactor during the PCR reaction. PCR buffer contains Tris and EDTA
which stabilizes the enzyme. dNTPs mixture containing ATP, GTP, CTP, TTP are
also added to make the new DNA strand.
The
PCR reaction is carried out in a machine called as thermocycler.
First,
the DNA duplex strand are separated by heating at 950C for 5
minutes.
The
PCR reaction takes place in three steps: -
- In the first step, the target DNA to be amplified is denatured at around 960C.
- The temperature is then reduced to around 500C to allow the annealing of the forward and reverse primers to the template DNA strand.
- The temperature is again increased to 720C to allow polymerization to occur using the dNTPs as the raw materials.
Final elongation
step which is also called as proof-reading step, is carried out at 720C
for 5-10 minutes.
At end of one
cycle, the target sequence on both the strands of template DNA has been copied.
The cycle is then repeated and theoretically 1 billion copies are generated
after 30 cycle.
2. Electrophoresis: -
Agarose
Gel Electrophoresis is used in
molecular biology to separate macromolecules such as DNA, RNA or proteins in a
matrix of agarose.
These
biomolecules are separated by applying an electric field to move the charged
molecules through the matrix. Since DNA is negatively charged, it will migrate
towards the positively charged anode.
Smaller
molecules travel faster than the larger molecules in the gel. This helps in
separating DNA (single, double stranded and even super-coiled) and RNA.
The separated
molecules can be viewed with strains that fluoresce under UV light (UV
trans-illuminator).
Polyacrylamide
Gel Electrophoresis of proteins is
mostly carried out in polyacrylamide gel under conditions that ensures the
dissociation of proteins into the individual polypeptide subunits.
These
subunits then migrate through the gel under the influence of electric field and
are separated based on their sizes. Smaller molecules travel faster than the
larger molecules in the gel.
The
effective range of separation of polyacrylamide gel depends upon the
concentration of polyacrylamide used to cast the gel and on the amount of
cross-linking.
3. Blotting: -
Blotting is
a technique used to identify different biomolecules after their separation on
electrophoresis. The molecule of interest is identified using either a labelled
probe i.e. radiolabelled complementary strand of nucleic acid or by using labelled
antibodies raised against a specific protein.
once the
complex of the labelled probe has been formed with the molecule of interest, it
is detected using the technique of autoradiography.
There are 4
types of blotting techniques i.e. Western blotting, Northern blotting, Western
blotting, eastern blotting.
4. Restriction digestion: -
The process
of cutting of DNA strand into smaller fragments at a specific site is called
restriction digestion. This is achieved by a group of enzymes known as “Restriction
endonucleases”. These enzymes cut the DNA at a particular site known as
restriction site.
Once the DNA
has been cut, the gene of interest can be inserted at that site. This is the
basis of recombinant DNA technology.
5. Ligation: -
Ligation is
a process of joining of two fragments of DNA together. This is carried out by
the help of enzyme called Ligases. Ligation is also an important step of
recombinant DNA technology as it allows the joining of gene of interest to
desired site in the host/plasmid.
6. Molecular cloning: -
Molecular
cloning can be defined as the process of introduction of new genes into a cell
or organism. In this technique, the gene producing the protein of interest is
isolated and inserted in a plasmid vector using restriction digestion and
ligation process.
This
so-called recombinant plasmid can then be inserted into bacterial or animal
cells for its expression.
Introduction
of this plasmid DNA in the bacterial cells can be achieved through several
ways: -
- Transformation: - the process of direct uptake of naked DNA by the bacterial cell from its surrounding environment.
- Conjugation: - the process of transfer of DNA through cell-cell contact. This is achieved by a specialized appendage called as pili. This type of gene transfer is called as horizontal gene transfer.
- Transduction: - the transfer of DNA using viral vector which infects the target cells.
Similarly,
there are several ways of transferring the recombinant plasmid DNA into the
eukaryotic cells. Few examples include: - transfection (by use of physical
or chemical means), electroporation, microinjection etc.
Molecular
cloning is one of the most important step of recombinant DNA technology. It is
used to study the effect of genes and their products on the organism. It can
also be used to create a mutant organism with different capabilities from its
parental species.
There are several other molecular biology techniques which are not listed above. All these techniques in conjugation with one another provide a very precise method to reveal the complexity of life.
Conclusion
Molecular biology is the study of
cellular mechanisms at molecular level. It provides scientists with a toolkit
to study the way life works.
Central dogma of molecular biology
explains the flow of information from store house i.e. DNA to the expressional
unit i.e. Proteins. The flow of information is always from DNA to RNA to proteins.
Every cell replicates its DNA during cell division. These replicated DNA is
then transcribed to produce RNA which in turn is used as template to synthesize
proteins.
Every cell follows the same principle
of central dogma. However, Retrovirus are exception to this principle.
Molecular biology shares relationship
with different biological sciences. The most related biological fields to
molecular biology are Biochemistry and Genetics.
There are many basic techniques used
in molecular biology. Few of them are PCR amplification, molecular cloning, Gel
electrophoresis, ligation, restriction digestion etc. there are several other
techniques which are used in conjugation by the researchers to reveal the
complexity of life.
Is there any basic technique which I missed out?
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