Gene cloning

Gene cloning is the process of generating genetically identical copies of a gene.

Gene cloning holds a significant position in the scientific arena. The reason for this is that we will be able to get rid of genetic diseases like sickle cell anemia and cystic fibrosis if scientists replace non-functional genes with the functional ones.

Principle of gene cloning

A desired sequence of DNA is inserted into a suitable vector, which results in a chimeric or recombinant DNA molecule. The vector transports the desired sequence of DNA into the host cell, usually a bacterium.

Within the host cell, the vector along with the gene it carries, replicates. Then the host cells divide and further replications of the vector take place. After multiple divisions, identical host cells are produced, usually called “a colony” or “a clone”.

Techniques

1. Recombinant DNA Technology

It is the technology that cuts and pastes the desired sequence of DNA together with the help of enzymes.

Requirements

Gene of interest

It is a particular section of the DNA strand that you desire to alter and replicate.

Molecular scissors

The restriction endonuclease enzyme acts as a molecular scissor that cuts as well as restricts the growth of the specific part of the viral DNA. The site at which it is cut is called the restriction site.

The endonuclease enzyme is extracted from a type of bacteria which consist of 400 enzymes. However, out of these, only 20 are used in biotechnology.

This enzyme was discovered by Hamilton O’Smith which was isolated from a bacterium named E.coli.

Molecular carrier

The molecular carrier is the vector that carries foreign DNA into the host DNA, resulting in the formation of recombinant, chimeric, or transgenic DNA.

 There are many molecular vectors such as plasmid, yeast, and lambda phage but the most important one is “plasmid”.

Plasmid holds such importance because it is an extra-chromosomal circular DNA that consists of many replication sites, resistance genes, and cloning genes.

Expression system

The most recommended expression systems are bacteria. They are treated with CaCl2 to increase their permeability, before introducing the Recombinant DNA.

Ways to isolate genes of interest

1. Isolate genes from chromosomes
2. Synthesize genes chemically in the laboratory
3. Synthesize genes from mRNA with the help of reverse transcriptase

Steps

Isolation

Dissolve the cell wall with the aid of enzymes and cut the desired segment of the DNA with the help of restriction endonuclease enzyme.

If the organism is a bacterium, then the enzyme required to dissolve its cell wall would be cellulase enzyme as its cell wall is composed of cellulose. 

On the other hand, the cell wall of fungi is made up of chitin. Therefore, the enzyme required to break its cell wall is the chitinase enzyme.

Separation

Separation of the desired fragments can be achieved through the process of gel electrophoresis.

Ligation

After this, join the foreign DNA and the vector DNA, forming recombinant DNA, with the help of ligase enzyme.

Transformation

In this step, transfer the recombinant DNA into the desired bacterial cell. 

Downstream Processing

Last but not the least; in downstream processing, you obtain the desired product.

2. Polymerase Chain Reaction (PCR)

PCR is an in-vitro method that generates thousands of millions of copies from a single or a few copies of genes, in a test tube.

With the help of this method, you can clone copies from any source. For example, from bacteria, viruses, animals, or plants.

Components

1. Template DNA
2. Primers
3. Deoxyribo-nucleoside tri-phosphate (dNTPs)
4. Taq polymerase (thermostable and withstands a temperature of 95°C)
5. Buffer
6. Co-factor (MgCl2)

Steps

Denaturation

2 strands of DNA are required. One side of the strand is 3` and the other is marked as the 5` end. Heat these strands at the temperature of 95°C for 1 minute. As a result of which, both the strands will be separated from each other.⁽¹⁾

Annealing

In annealing, the strands need to be cooled down at the temperature of 55°C, for no more than 2 minutes. Then add a primer at the 3` end of the DNA.⁽¹⁾

Extension

In this process, add Taq polymerase at the temperature of 72°C, for 1.5 minutes. This enzyme begins the extension of the strand from the 3` end.⁽¹⁾

Conclusion

The cycle ends with these 3 steps. With the completion of one complete cycle, 2 copies are obtained. At the end of the 2^nd cycle, 4 copies are achieved and this process goes on until thousands of millions of copies are obtained.

At the end of the 32^nd cycle, a million folds of the target DNA strands are achieved.⁽⁴⁾

Application of gene cloning

• It is used to detect certain infectious agents such as HIV, HBV, and HCV, etc. 
• It can also help discover various micro-organisms that may be present in water or food.
• It is also used in DNA fingerprinting.
• It can detect the genetic mutations that are held responsible for multiple genetic disorders like color blindness, hemophilia, and leukemia, etc.
• It is also used in criminology. It helps the investigators to find criminals by matching the DNA samples found at the site of crime and the DNA samples of the suspects.
• It is also used to synthesize useful products like hormones, vitamins, and antibiotics, etc.

Advantages and disadvantages of cloning

Advantages	Disadvantages
Helps infertile couples have kids	Decreases the genetic diversity as the offsprings are the same copies of their parents
Plays an incredible role in organ replacement	There exist many side effects in cloning that are yet to be studied, making it a risky process
Helps cure diseases	May produce new diseases
Through cloning, amazing individuals such as Newton and Einstein can be formed	It can lead to further divisions as the cloned human may not feel “human” enough

Frequently Asked Questions

References

1. https://pubmed.ncbi.nlm.nih.gov/21400259/
2. https://medlineplus.gov/cloning.html
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1120620/
4. U Satyanarayan – Text book of Biochemistry
5. Lippincott – Textbook of Biochemistry