Tools used in Recombinant DNA Technology
Introducing the desired gene into the genome of a host organism is a challenging process. It involves insertion of the selected gene into the host which is facilitated by a vector which helps integration of the gene to form the recombinant DNA. This DNA is then introduced inside the host, sustained and carried forward.
Cloning Vectors
Cloning vector refers to a minute DNA molecule which self-replicates itself inside the host cell. Cloning vectors are utilized for duplicating the desired DNA fragment into the host cell. Features of a cloning vectors are:
They should be small in size.
They should be self-replicating inside the host cell.
They should have a restriction site for action of the Restriction Endonuclease enzymes.
Insertion of a benefactor DNA fragment should not hinder the replication of the vector.
They should possess some marker gene to identify the recombinant DNA.
They should have multiple cloning sites.
Recombinant DNA Technology
Recombinant DNA technology changes the phenotype of an organism (host) with the help of a genetically transformed vector. The cloning vector is then inserted into the genome of the organism. The process involves the insertion of a desirable foreign DNA with the gene of interest into the genome of the host. This gene is called recombinant gene and this method or technique is called recombinant DNA technology. A recombinant DNA technology can be carried out and accomplished with the support of some fundamental tools. The different tools used for the function are mentioned below:
Tools of Recombinant DNA technology
Restriction Enzymes:
These are enzymes that have restriction endonucleases that help in cutting, polymerases that aid in the synthesis of DNA, and ligases that facilitate binding.
The restriction endonucleases used in recombinant DNA technology have a vital role in outlining the location at which the desired gene of interest is introduced into the vector genome.
These are of two types usually, named endonucleases and exonucleases. The endonucleases cut within the strand of DNA whereas the exo-nucleotides are involved in cutting the ends of the DNA.
The restriction endonucleases are specific to palindromic sequences and cut the DNA at specific points. There are 3 chief types of restriction endonuclease enzymes: Type-I Restriction Endonucleases, Type-II Restriction Endonucleases, and Type-III Restriction Endonucleases.
They inspect the length of DNA and cut at the specific site known as the restriction site. This creates sticky ends in the sequence.
The gene of interest and the vectors are cut by the same restriction enzymes to acquire the corresponding sticky ends, after which ligases help in binding the sticky ends.
Vectors
The integration of the gene of interest is done with the help of vectors. These are the vehicles that drive the gene inside the host organism. The most commonly used vectors are bacteriophages and plasmids for their high copy number.
Host Organism
The organism in which the recombinant DNA is introduced is called the host organism. It is the ultimate tool into which the vector drives the gene of interest using the enzymes.
Techniques like microinjection, gene gun, biolistic are employed to insert the recombinant DNA within the organism. This can be also carried out using alternate heating and cooling or the use of calcium ions.
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Steps of Recombinant DNA Technology
Selection and seclusion of DNA to be inserted.
Selection of an appropriate cloning vector.
Insertion of DNA into the vector to structure recombinant DNA molecules.
Recombinant DNA molecule is launched into a suitable host.
Selection of altered host cells.
Articulation and proliferation of DNA inserted in the host
DNA is a common term that the majority of students might have heard or read in different textbooks or in their surroundings, but what does DNA stand for and what is its purpose and functions in the field of science. DNA stands for Deoxyribonucleic acid which is a scientific term given to it, DNA in simple language is a molecule that contains all the genetic information of an organism.
Here, genetic information can be referred to as the information of ancestors of an organism. Now the question is what type of information this could be, this information could be anything related to as simple as the complexion of the organism, physical body alignments like face curves, eyes, hair colour, nose dimensions, height or it could also be internal information inheritance like emotions, personality, etc.
Role Of DNA And Tools Of Recombinant DNA Technology
DNA is a topic that covers a huge wide area of topics in the field of bio and is a vast and very deep topic. Scientists are still unable to fully learn the working of DNA and its working in organisms and the work is still going on to uncover the deep knowledge of DNA. Along with this the scientists are also working on DNA manipulation or alterations in DNA which have emerged as a very beneficial and helpful method majorly in the medical field and much more. This method of altering or modifying the DNA is termed Recombinant DNA technology. Developing recombinant DNA is not a simple task as it includes different processes and various tools of recombinant DNA technology.
FAQs on Tools of Recombinant DNA Technology
1. What are the essential tools required for carrying out recombinant DNA technology?
To perform recombinant DNA technology, a specific set of biological tools is essential. The main tools include:
- Restriction Enzymes: Used to cut DNA at specific sites, often called 'molecular scissors'.
- Cloning Vectors: These act as vehicles to carry the desired foreign DNA into a host cell. Plasmids are a common example.
- DNA Ligase: This enzyme acts as 'molecular glue' to join the DNA fragments together.
- Host Organism: A living cell, usually a bacterium like E. coli, into which the recombinant DNA is introduced for cloning.
2. What is the specific role of restriction enzymes in this process?
Restriction enzymes play a crucial role by acting as precise cutting tools. Each enzyme recognises a specific, short sequence of DNA known as a recognition site. It then cuts the DNA strand at that site, often creating 'sticky ends'. These sticky ends are short, single-stranded overhangs that can easily pair with complementary sticky ends on another DNA fragment, allowing the gene of interest to be inserted into a vector.
3. Why is a 'cloning vector' necessary in genetic engineering?
A cloning vector is necessary because a foreign piece of DNA cannot replicate on its own inside a host cell. The vector acts as a delivery vehicle that carries the desired gene into the host. Once inside, the vector uses the host's cellular machinery to replicate, making many copies of itself and the inserted gene along with it. Plasmids and bacteriophages are common examples of vectors.
4. What key features make a plasmid a good cloning vector?
A plasmid is considered a good cloning vector because it possesses several essential features:
- Origin of Replication (ori): This is a DNA sequence that allows the plasmid to replicate independently within the host cell, creating multiple copies of the desired gene.
- Selectable Marker: A gene, such as one for antibiotic resistance, that helps identify which host cells have successfully taken up the vector.
- Cloning Sites: One or more unique recognition sites where restriction enzymes can cut the plasmid to insert the foreign DNA.
5. How does the enzyme DNA ligase function to create a recombinant DNA molecule?
DNA ligase acts as a molecular glue. After a restriction enzyme cuts both the vector DNA and the gene of interest to create complementary 'sticky ends', these ends pair up through hydrogen bonds. However, this connection is weak. DNA ligase then creates strong, permanent phosphodiester bonds between the sugar-phosphate backbones of the vector and the inserted DNA fragment, sealing them together to form a stable recombinant DNA molecule.
6. How do scientists identify the host cells that have been successfully transformed with the recombinant DNA?
Scientists use a tool called a selectable marker, which is part of the cloning vector. For example, if the vector carries a gene for ampicillin resistance, the host cells are grown on a medium containing ampicillin. Only the cells that have successfully taken up the vector will survive and grow, because they now have the resistance gene. The untransformed cells will die, making it easy to select the right ones.
7. What are some real-world applications of using these genetic tools?
The tools of recombinant DNA technology have many important applications across various fields. For example, they are used to:
- Produce therapeutic proteins like human insulin for treating diabetes and human growth hormone.
- Develop genetically modified (GM) crops, such as Bt-cotton, which is resistant to pests.
- Create vaccines and diagnose diseases.
- Facilitate gene therapy to correct genetic disorders.
8. Why is it important that restriction enzymes are so specific about where they cut DNA?
The high specificity of restriction enzymes is critical for the success of genetic engineering. Each enzyme recognises a unique DNA sequence. This precision ensures that the DNA is cut at predictable and desired locations, allowing scientists to isolate a specific gene and insert it correctly into a vector. If the enzymes were not specific, they would cut the DNA randomly, making it impossible to control the process and create a functional recombinant molecule.
