Recombinant DNA Technology Steps, Application, Tools, and Limitations
Recombinant DNA Technology Steps, Application, Tools, and Limitations

Molecular biology

Recombinant DNA Technology Steps, Application, Tools, and Limitations

Table of Contents show 1 What is Recombinant DNA Technology? 2 Tools Of Recombinant DNA Technology 3 Steps of Genetic Recombination Technology...

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MN Editors
This article writter by MN Editors on January 05, 2022

Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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What is Recombinant DNA Technology?

The process of creating artificial DNA by the mixing of various genetic substances (DNA) from various sources is known as Recombinant DNA technology. This technology has become commonly described as genetic engineering.

Recombinant DNA technology was first discovered from an investigation into restriction enzymes in years 1968, through Swiss microbiologist Werner Arber,

Inserting the desired gene in the host’s genome isn’t as easy as it seems. It requires the selection of the gene you want to administer into the host . This is followed by the selection of the best vector which the gene needs to be integrated, and the formation of recombinant DNA.

So, the recombinant DNA is been introduced to the host. Then it must remain within the host and passed through to offspring.

Recombinant DNA Technology
Recombinant DNA Technology

Tools Of Recombinant DNA Technology

The enzymes that comprise the restriction enzymes aid in cutting the polymerases, they aid in the production of synthesized proteins, and the ligaseshelp to connect. The restriction enzymes that are used in Recombinant DNA technology play a significant role in determining the place where the desired gene will be inserted into the genome of the vector. There are two kinds of them of restriction enzymes, which are Endonucleases as well as Exonucleases.

The Endonucleases cut through the DNA strand , while the Exonucleases eliminate nucleotides from both ends. The restriction endonucleases are specific to sequences that typically are palindrome sequences that cut DNA at specific places. They examine how long DNA is and then make cuts at a specific location, which is known as”the restriction” site. This results in sticky ends within the sequence. The desired genes as well as their vectors will be cut with similar restriction enzymes that produce the sticky notes in the opposite direction and make the job of ligases straightforward to attach that desired gene with the appropriate vector.

The vectors assist in carrying and connecting the desired gene. They are a crucial component of the tools used in Recombinant DNA technology since they are the most powerful vehicles to carry forward the desired gene into the host. Plasmids and bacteriophages are among the most frequently used vectors used in the field of recombinant technology and are employed because they have an extremely high copy number. The vectors consist of the replication’s origin. It is a sequence of nucleotides that are the place where replication begins and a marker that can be selected are genes that exhibit resistance to specific antibiotics, such as ampicillin and cloning spots that are identified by the restriction enzymes that are where the desired DNAs are put in.

Host organisms – where the recombinant genome is introduced. Host is the most powerful device of recombinant DNA technology that takes in the engineered DNA vector that has been crafted with the desired DNA, with the aid of enzymes.

There are many methods by which these DNA recombinants are introduced into the body, including microinjection, biolistics, gene gun alternate heating and cooling, the using calcium ions and so on.

Steps of Genetic Recombination Technology

Steps of Genetic Recombination Technology
Steps of Genetic Recombination Technology

1. Isolation of Genetic Material

The initial stage in rDNA technique is to separate that desired DNA from its original form i.e. completely free of other macromolecules. DNA is found in the cell membrane with other macromolecules, such as polysaccharides (RNA), polysaccharides as well as lipids, DNA needs to be separated from and purified. This requires enzymes such as cellsulase and lysozymes as well as proteases like ribonuclease and proteases. Other macromolecules can be removed using other treatments or enzymes. In the end, the presence of ethanol causes DNA to separate into fine threads. This is then spun out to create pure DNA.

2. Restriction Enzyme Digestion

Restriction enzymes function like molecular scissors which cut DNA at certain locations. These reactions are known as’restriction enzyme digestions’. They involve incubating the DNA purified with the specific restriction enzyme at the conditions that are optimal for the enzyme in question. The method ‘Agarose Gel Electrophoresis shows the progression of digestion using the restriction enzyme. This method involves separating the DNA using the agarose gel. When current is applied the DNA that is negatively charged moves through the electrodes to the positive, and is separated out according to size. This permits cutting out DNA fragments that have been digested. The DNA from the vector is processed with the same method.

3. Amplification Using PCR

Polymerase Chain Reaction, also known as PCR is a technique for creating multiple copies of DNA sequences with the help of DNA Polymerase, in vitro. It allows you to multiply one copy or couple of copies of DNA into millions to thousands of copies. PCR reactions are performed by ‘thermal cycles’ that use the following elements:

  • Template – DNA to be amplified
  • Primers – small, chemically synthesized oligonucleotides that are complementary to a region of the DNA.
  • Enzyme – DNA polymerase
  • Nucleotides – needed to extend the primers by the enzyme.

The DNA fragments that have been cut can be amplified with PCR before being ligated to cutting vector.

4. Ligation of DNA Molecules

The purified DNA as well as the interest vector are cut using this same enzyme. This results in the DNA fragment that has been cut and the cut vector which is now unlocked. The method of joining these two pieces with the enzyme ‘DNA Ligase”‘ is called ligation. The resultant DNA molecule will be an amalgamation made up of two DNA molecules: both the interest and vector. In the field of genetics, the intermixing of multiple DNA strands is referred to as the process of recombination. Therefore, this hybrid DNA molecule can also be called a recombinant DNA mole and is also called the recombinant DNA technique.

5. Insertion of Recombinant DNA Into Host

In this process the recombinant DNA is transferred into a host cell. It is usually bacteria cells. This is known as “Transformation”. Bacterial cells cannot readily accept DNA from other species. So, they undergo treatment to be ‘competent’ for accepting new DNA. The methods used could be thermoshock, CaIon Treatment, Electroporation, etc.

6. Isolation of Recombinant Cells

The transformation process creates the transformation process to create a mixed number of transformed and non-transformed host cells. The process of selection involves filtering transformed host cells to the extent that they are. To distinguish recombinant cells from non-recombinant cells the marker gene in the vector plasmid is used. For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene. When pst1RE is utilized, it eliminates Ampicillin resistance gene in the plasmid in order for the recombinant cells to becomes susceptible to Ampicillin.

Application of Recombinant DNA technology

  • Recombinant DNA is extensively used in medicine, biotechnology and research.
  • The most widely-used application of DNA recombinant is in basic research, and the technology is crucial to the majority of current research in biomedical and biological sciences.
  • Recombinant DNA can be used to determine, map the sequence of genes as well as to identify their functions.
  • Recombinant proteins are used extensively in laboratory experiments as reagents as well as to make antibodies to study the synthesis of proteins in cells and in organisms.

A variety of other applications for Recombinant DNA can be found in food production, industry Human and veterinarian medicine, agriculture and bioengineering.

  • Recombinant chymosins: Recombinant chymosinis found in rennet, chymosin can be described as an enzyme that is required for the production of cheese. This was the very first food additive that was genetically engineered and that was commercially used. Traditionally, processors sourced the chymosin ingredient from rennet which is a product derived from the stomach of the fourth calves that are fed milk. Scientists have engineered an unpathogenic kind of strain (K-12) from E. E. coli to allow large-scale lab production of this enzyme. The is a microbiologically produced recombinant enzyme that is similar in structure to the calf-derived enzyme, is less expensive and is manufactured in huge quantities.
  • Recombinant human insulin: Recombinant human insulin has almost entirely substituted insulin from animals (e.g. cattle and pigs) to treat insulin-dependent diabetes. A range of different insulin preparations made from recombinant sources are widely used. Recombinant insulin is produced through the introduction of human insulin genes into E. coli, or yeast (Saccharomyces cerevisiae) which produces insulin that is suitable for human consumption.
  • Human growth hormone recombinan: Human growth hormone recombinant (HGH, Somatotropin) is administered to patients whose pituitary glands produce insufficient amounts to allow the normal development and growth. Prior to the time that the recombinant HGH was made readily available HGH to treat ailments was obtained from the pituitary glands from cadavers. This unwise practice resulted in the development of CreutzfeldtJakob disease in a few patients. Recombinant HGH has eliminated this issue and is utilized to treat patients.
  • Recombinant blood-clotting factor VIII: A protein used to clot blood. It is given to patients suffering from types of hemophilia bleeding disorder who are not able to produce factor VIII in sufficient quantities to ensure the normal process of blood coagulation. Prior to the creation of recombinant factor VIII the protein was derived by processing large amounts of blood donated by multiple people which carried a significant chance of transmission of blood-borne infections, such as HIV and hepatitis B. The entry in DrugBank.
  • Recombinant hepatitis B vaccine: Hepatitis B infection is prevented by using the reconjugation hepatitis B vaccine. It is a type of hepatitis B surface antigen of the virus that is created in yeast cells.
  • Diagnosis of infection with HIV: The diagnosis of an infection caused by HIV The three most commonly used methods to diagnose HIV disease has been created with the recombinant DNA. The test for antibodies (ELISA also known as western blot) utilizes an Recombinant HIV protein to determine any antibodies the body produces in response to HIV infection.
  • Golden rice: A recombinant type of rice engineered to express enzymes involved in the biosynthesis of b-carotene.
  • Herbicide-resistant plants: Commercial varieties of key agriculture crops have been developed which contain a recombinant genetic that confers resistance the herbicide called glyphosate (trade trade name Roundup) and makes it easier to manage the control of weeds through glyphosate application.
  • Insect-resistant crops: Bacillus thuringeiensis is a bacteria which naturally produces an enzyme (Bt toxin) that has insecticidal properties. The bacterium has been added to crops to control insects as a method for years and is widely used in gardening and agriculture. Recently, plants were created that have a recombinant expression of the protein bacterial that can effectively control certain insects. Environmental concerns associated with the transgenic crops are not completely addressed.

Limitations of Recombinant DNA technology

  • The destruction of native species within the habitat that genetically altered species are introduced into.
  • Resilient plants could in theory give rise to resistant weeds that can be difficult to manage.
  • Migration of DNA that is proprietary between different organisms.
  • Recombinant organisms are contaminating the natural ecosystem.
  • The recombinant organisms consist of clones, which are susceptible to disease in the exact same way. One single pest or disease can decimate the entire population in a short time.
  • Superbugs are believed to be the cause of the superbug.
  • Concerns about the ethical implications of humans trying try to be God and interfere with natural process of choosing. This is exacerbated due to the fear of what can be made with the help of technology and how it is going to affect our civilization.
  • A system like this could cause people to have their genetic data stolen and used without their permission.
  • Many are concerned about the security of altering foods and medications with recombinant DNA.
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