What is Molecular biology?

Molecular biology is the study of cellular mechanisms at molecular level. This means that, molecular biology is focused on understanding the process of replication, transcription and translation of genetic materials.

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. 

1. They may use it to study the effect of single gene or protein on the organism. 
2. They may also study the effect of mutation in a single gene or protein.
3. 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.

1. 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.
   
2. 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.
3. 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.

1. 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: - 

1. In the first step, the target DNA to be amplified is denatured at around 96⁰C.
2. The temperature is then reduced to around 50⁰C to allow the annealing of the forward and reverse primers to the template DNA strand.
3. 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). 

Jennifer0328 [CC BY-SA]

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. 

CNX OpenStax [CC BY]

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?