Tools of Recombinant DNA Technology

Biotechnology

The name “biotechnology” comes from the Greek words “bios,” which means “life,” “techno,” which means “technology,” and “logos,” which means “language,” or “proof.” Biotechnology is the technical use of living organisms for a variety of purposes, including food, medicine, medicines, and recycling. Biotechnology is the combination of biology and technology for human benefit and sustainable development. Biotechnology is a technology that uses biological systems, living organisms, or parts of them, to produce or create various products....

Principles of Biotechnology

Among many, the two fundamental techniques that  contributed to the development of  modern biotechnology are :...

Recombinant DNA

This restriction is overridden by genetic engineering techniques such as recombinant DNA production, gene cloning, and gene transfer, which enable us to extract and introduce only one or a small number of desired genes without introducing unwanted genes into the desired organism....

DNA Cloning

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Origin of Replication

The origin of replication is a specific DNA sequence in the chromosome that is responsible for initiating replication. Consequently, for any foreign piece of DNA to multiply in an organism, it must be a component of a chromosome(s) with a certain specific sequence referred to as the ‘origin of replication’.The genomic regions where DNA replication begins are commonly referred to as DNA replication origins. However, they do have at least two distinct elements: the DNA region that will form the pre-replication complex and that will be recognized and bound by specialized proteins....

Plasmids

Plasmids are small, circular DNA molecules that can reproduce autonomously, as they do not depend on an organism’s chromosomal DNA for replication. As a result, plasmids are often referred to as extrachromosomal DNA. Plasmids are important genetic engineering tools as they help in gene cloning and gene therapy....

Recombinant DNA Technology

Recombinant DNA technology modifies the phenotype of an organism (the host) by using a genetically modified vector. This cloning vector is injected and incorporated into the organism’s genome....

Tools of Recombinant DNA Technology

The following is a discussion of the various tools used for various purposes. The above explanation has shown us that key tools such as restriction enzymes, polymerase enzymes, ligases, vectors, and host organisms are essential for genetic engineering or recombinant DNA technology to be successful....

Restriction Enzymes

The position at which the desired gene is inserted into the vector genome is greatly influenced by the restriction enzymes utilized in recombinant DNA technology. E-coli was the first restriction enzyme to be isolated in 1963 from the development of bacteriophage in the laboratory....

Separation and isolation of DNA fragments

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Vectors

The vectors support carrying and inserting the desired gene. These are a very important part of the tools of recombinant DNA technology as they are the final vehicles that carry the desired gene further into the host organism. The two most popular types of vectors used in recombinant DNA technology are bacteriophages and plasmids....

Cloning Vector

A vector is a DNA molecule used to introduce foreign DNA into a host cell. It is capable to self-replicate and integrating into the host cell. These vectors have aided in the analysis of the molecular structure of DNA....

Types of Cloning Vectors

Plasmid: The most used bacterial cloning vectors are plasmids. These cloning vectors have a site that enables the insertion of DNA fragments, such as a polylinker with numerous commonly used restriction sites that can bind DNA fragments. A plasmid is 4361 bp in size, and its cloning range is 0.1 to 10 kb. While the gene is still isolated from E. coli, it is ampicillin- and tetracycline-resistant. Examine PBR322 Bacterial Artificial Chromosome: A bacterial artificial chromosome (BAC) is a DNA molecule that has been produced and is used to clone DNA sequences in bacterial cells (for example, E. coli). BACs are frequently utilized in the context of DNA sequencing. The BAC is 11827 bp in size, and the cloning range is 35-300 kb. Chloramphenicol and lactose metabolizing genes are the marker genes. It is artificially synthesized by f-plasmid modification. For instance, pUvBBAC Yeast Artificial Chromosome: Yeast artificial chromosome (YAC) is a DNA molecule designed by humans that is used to clone DNA sequences in yeast cells. YACs are frequently utilized in the mapping and sequencing of genomes.  YAC is 11400 bp in length, while the cloning vector is 100-1000 kb in size. The marker is comparable to yeast’s. It is synthetic and contains a yeast centromere obtained from Saccharomyces cerevisiae. Bacteriophage: Bacteriophage lambda is utilized as an expression vector in the cloning procedure. It has the ability to replicate to 24kbp size of DNA which is higher than plasmids. It has a size of 48502 bp, of which one-third is unnecessary. It can only recombinant approximately 4-5 kbp of donor DNA. Lambda genome is one such example. Cosmid: Cosmid has a size of 7900 bp and a cloning limit of 30-50 kb. It consists of similar characteristics to both phase and plasmid. for Example-super COS1 Human Artificial Chromosome: It is an artificial chromosome that is utilized to transfer human genes and has no restriction on cloning as it can carry a large portion of the DNA....

Process of gene cloning

This involves isolating the gene from the donor cell, integrating it into a small carrier molecule (also called a vector), and then replicating it into the host cell. A few steps in this process are mentioned below....

Process of Recombinant DNA Technology

Recombinant DNA technology consists of selecting the appropriate gene for administration into the host, after this, the correct vector is selected with which the gene is to be integrated and hence the recombinant DNA is formed. This recombinant DNA is then to be introduced into the host. Finally, it must be preserved in the host and passed on to the progeny....

Insertion of recombinant DNA into the host

The host is the ultimate tool of rDNA technology, which absorbs the vector engineered with the desired DNA with the help of enzymes. There are several techniques to introduce the desired recombinant DNA into the host organism. some important techniques such as Microinjection, different heating and cooling methods, use of the gene cannon, biolistics, etc....

Obtaining the Foreign Gene Product

In recombinant technologies, the desired gene is selected, followed by a selection of an ideal vector in which the specific gene is integrated, and then the gene of interest is fused with the vector to form recombinant DNA. Once this foreign DNA is integrated, the host multiplies and eventually produces the desired protein. The rDNA must be retained in the host and passed on to the progeny. In order for the desired protein to be produced, the gene that encodes for it needs to be expressed. It occurs only under optimized conditions. Not only the target protein has to be expressed but it has to be produced on a large scale....

Bioreactor

A bioreactor supports to the production of a large quantity of culture. A bioreactor is a large vessel where various cells, including human or plant, or animal cells, can be cultured to produce new biological products. It provides appropriate conditions such as temperature, pH, substrate, oxygen, and so on that are necessary for the culturing of cells to develop desired products. The two types of bioreactors utilized for this purpose are simple stirred-tank bioreactors and sparged stirred-tank bioreactors....

Downstream Processing

Downstream processing is a sequential phase in which the final products are isolated, purified, and preserved before they are marketed. In this phase, the final product is developed with additives such as preservatives, pigments, and so on....

FAQs on Biotechnology: Principles and Processes

Q1: What are the most essential methods in biotechnology?...