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Recombinant DNA Technology

A Special Project in

Advanced Cellular and Molecular Biology-

Genetics

Submitted by:

MARY LYNDIE G. BORGONIA, LPT

Student, MA-Biology

Presented to:

DR. JUNNASIR SAKILAN

Instructor

June 2022
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Table of Content

Table of Content

Cover Page 1

Outline 2

I- Introduction 3

II- Background of the Process 6

III- Methodology 8

IV- Conclusion 14

V- References 15
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I. Introduction

DNA was the most important biological breakthrough of the twentieth century. Its

discovery has increased our ability to diagnose disease, determine genetic predisposition to

disease, develop novel treatments to treat disease, apply gene therapy as a treatment, and

construct "custom drugs" based on individual genetic profiles in modern medicine and genetic

research according to Gaxhi (n.d.). Since then, different DNA technologies emerged including

Recombinant DNA Technology (rDNA Technology). As stated by Salave (2022), recombinant

DNA technology is the art and science of connecting or merging two or more DNA molecules

from various plant, animal, or microorganism species. This technology is utilized to improve

the quality of existing genetic combinations that are of value to science, medicine, agriculture,

and industry.

The work of American biochemists Stanley N. Cohen, Herbert W. Boyer, and Paul Berg

was crucial in the development of recombinant DNA technology. Berg conducted the first

successful gene-splicing experiment in the early 1970s, combining DNA from two distinct

viruses to create a recombinant DNA molecule. Boyer and Cohen then proceeded to insert

recombinant DNA molecules into bacteria, which reproduced and produced numerous copies

of the recombinant molecule. Boyer and Cohen went on to develop recombinant plasmid

generation techniques (Pham, 2018). Boyer co-founded Genentech with Robert A. Swanson in

1976, which marketed Boyer and Cohen's recombinant DNA technology (Griffiths, 2020). As

stated by Salave (2022), this procedure is also known as genetic engineering because it

improves an organism's overall superiority.

Werner Arber discovered restriction enzymes in bacteria that digest invading viral DNA

molecules, which sparked the original idea for recombinant DNA technology. Geneticists

learned to "cut" and "paste" DNA molecules as a result of this discovery, and novel restriction
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enzymes for cutting and pasting were discovered or manufactured. Stanley Cohen and Herbert

Boyer collaborated in 1972 to progress the development of recombinant DNA technology. In

1976, they founded Genentech, the first firm dedicated to recombinant DNA technology as

revealed by Pham.

Laboratory geneticists' primary purpose is to extract, define, and modify genes because

the genes is the focus of genetics as revealed by Griffiths (2020). Although isolating a sample

of DNA from a group of cells is very simple, locating a specific gene within this DNA sample

is comparable to finding a needle in a haystack. Consider that each human cell has about 2

meters (6 feet) of DNA. As a result, a tiny tissue sample can contain n kilometres of DNA.

However, researchers may now isolate one gene or any other section of DNA and ascertain its

nucleotide sequence, investigate its transcripts, alter it in highly specific ways, and reintroduce

the modified sequence into a living organism using recombinant DNA technology as stated by

Griffiths.

Pham (2018) mentioned that recombinant DNA technology is a powerful DNA-based

technique that brings in a new phase in biotechnology. A gene or numerous genes can be

identified, cut, and inserted into the genome of another organism using this approach. The

earliest medical biotechnology medications and its most significant breakthrough, the human

insulin, was created using this technology.

The process of Recombinant DNA Technology involves six processes as indicated by

Salave: (1) Isolation of Donor or Foreign DNA; (2) Digestion of donor or foreign DNA (3)

Introduction of target DNA into vector to create recombinant DNA molecule, (4) DNA

Molecules Ligation, (5) PCR-amplification of gene copies, and (6) gene insertion into host

organisms.
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The existence of rDNA Technology has a wide range of beneficial applications in

several fields. Recombinant DNA technology has prompted the development of a wide range

of biopharmaceutical products, including recombinant proteins that provide major

advancements in treating a wide range of medical diseases and conditions (Pham, Yilma, Feliz,

Majid, Mafferone, Walker . . . Yoon, 2019). Some vaccines and other agricultural products also

use rDNA Technology. Recent breakthroughs in recombinant DNA technology, microbial

genetics, and microbiology have the potential to improve the efficiency and profitability of

existing bioprocesses.
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II. Background of the Process

Unlike traditional approaches to overcoming agriculture, health, and environmental

issues through breeding, traditional medicines, and pollutants degradation through

conventional techniques, genetic engineering makes use of modern tools and approaches, such

as molecular cloning and transformation, which take less time and produce more reliable results

as stated by Shinde, Chavhan, Sapkal & Shrikhande (2016). Several reasons, such as food

scarcity leading to hunger, various types of deadly diseases, environmental issues resulting

from rapid industrialization and urbanization, and many others, put human future at risk.

Traditional strategies have been superseded by genetic engineering, which has a better capacity

to overcome such problems (Khan, Ullah, Siddique, Nabi, Manan, Yousaf, & Hou, 2016)

Biological tools include enzyme, vector, and host in the conduct of a recombinant DNA

technology. The enzymes will aid in the cutting (restriction enzymes), synthesizing

(polymerases), and binding of DNA (ligases). In this technique, restriction enzymes play a

crucial role. The restriction enzyme will cut at a specific restriction site inside the DNA

molecule. In most cases, the restriction enzyme will leave sticky ends in the DNA sequence to

aid in binding to the desired gene.

The desired gene will be carried by the vector. Recombinant DNA technique relies

heavily on vectors. They are the last vehicles for delivering genes of interest into the host

organism. To date, several forms of vectors have been created; nevertheless, plasmids and

bacteriophages are the most often utilized vectors. A vector must have the same restriction sites

as the target. The required gene to make gene integration easier Vectors should also contain

selection and identification sequences, such as an ampicillin resistance gene and cloning sites.

The cell into which the recombinant DNA is injected is known as the host organism.

Bacteria, fungi, and mammal cells have all served as hosts so far. Microinjection, biolistics,
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gene gun, alternate cooling and heating, and calcium phosphate ions have all been utilized to

introduce vectors into hosts.

Bioengineered or genetically modified organisms are created by transforming cells

using rDNA (GMOs). The GMOs have acquired new characteristics from another organism as

revealed by Suza and Lee (2021). Vaccines and insulin, are two of the many products that come

from rDNA technology.

Fig.1. examples recombinant DNA strategies for vaccine

development
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III. Methodology

Methods of Recombinant DNA Technology

Selection and isolation of

DNA

Digestion of donor/foreign
DNA (Cutting)

Introduction of target DNA

into vector to create
recombinant DNA molecule

DNA molecules Ligation

Amplification

Gene insertion into host

organisms.

Fig.2. the steps in conducting Recombinant DNA Technology

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1. Selection and Isolation of DNA

https://courses.lumenlearning.com/wm-
biology1/chapter/reading-biotechnology-and-genetic-
information/

1.1. It is the initial step in recombinant DNA (rDNA) technology, in which ethanol is used to

separate a desired gene-containing DNA fragment from other macromolecules such as RNA,

polysaccharides, proteins, and lipids. Isolating cells allows for the crude isolation of donor

(foreign) DNA. Detergents break lipid membranes, phenol or proteases destroy proteins,

RNase degrades RNAs, and DNA remains at the end.

1.2. Crude isolation of plasmid vector DNA is accomplished by an alkaline lysis procedure or

by boiling cells which removes bacterial chromosomal DNA from plasmid DNA.

1.3. To get purer DNA from either (1) or (2), crude DNA is a) Fractionated on a CsCl2 gradient

b) Precipitated with ethanol c) Poured over a resin column that specifically binds DNA
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2. Digestion of donor/foreign DNA (Cutting)

https://microbeonline.com/agarose-gel-electrophoresis/

In the presence of specific temperature, concentration, and duration, restriction

enzymes are utilized to cut the foreign DNA fragment at a specified site/location. The foreign

DNA fragments are separated using the Agarose Gel Electrophoresis method.

2.1.Mechanical shearing can cut DNA into huge bits.

2.2.Restriction enzymes are the molecular genetics scissors. Restriction Endonucleases

are enzymes that detect certain nucleotide sequences in DNA and break the DNA

chain at those locations. RE has been isolated in several forms and is now

commercially available. The majority of them cut at certain palindromic DNA

locations (sequence that is the same on both antiparallel DNA strands). These cuts

can be staggered to produce "sticky or overhanging ends" or blunt to produce flush

ends.
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3. Introduction of target DNA into vector to create recombinant DNA molecule

https://www.nature.com/scitable/topicpage/the-biotechnology-revolution-pcr-and-the-use-553/

The donor and vector DNAs must be linked together after they have been extracted

and cut. A foreign DNA fragment is joined to vector DNA that is suitable

molecule using the polymerase chain reaction (PCR) method. The DNAs are mixed

together in a tube. If both have been cut with the same RE, the ends will match up

because they are sticky.

4. DNA Molecules Ligation

https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-
cloning-tutorial/a/restriction-enzymes-dna-ligase

DNA ligase is the glue of molecular genetics that holds the ends of the DNAs

together. DNA ligase creates a phosophodiester bond between two DNA ends. The

ligation process is carried out in the presence of DNA ligase enzyme. By uniting
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the two free cohesive ends of the foreign DNA fragment, a circular, double-stranded

recombinant DNA molecule is created (along with the vector DNA molecule).

5. Amplification

Using the DNA polymerase enzyme, the same foreign DNA molecule is

replicated, cloned, and amplified to generate many copies. It is necessary to amplify

the recombinant DNA molecule in order to extract significant amounts of it. The

recombinant DNA is transformed into a bacterial host strain to do this. (The cells

are treated with CaCl2, DNA is added, the cells are heat shocked at 42 ℃, and DNA

enters the cell. When the cell divides, the recombinant molecules are duplicated in

both daughter cells, which will divide later. The DNA is thus amplified.

6. Gene insertion into host organism

Bacterial transformation. https://www.khanacademy.org/science/biology/biotech-dna-

technology/dna-cloning-tutorial/a/bacterial-transformation-selection
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Transformation is the process of inserting a recombinant DNA molecule into

the genome of a recipient or host bacterium. Foreign DNA fragments are not readily

accepted by the host bacterial cells. For effective transformation, such host bacterial

cells are given thermal shock, Ca+ ion therapy, electroporation treatment.

The isolation, identification, and separation of the transformed or recombinant

DNA molecule from the host bacterium genome are all included in this step. The

gene marker of the plasmid vector is used in this process.

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IV. Conclusion

The more things in the world that are found and brought to light, the more progress is

made. In biology, recombinant DNA technology is a critical research tool. It enables scientists

to modify DNA fragments in the laboratory in order to examine them. This approach can be

used to join (or splice) DNA from different species or to create new genes.

Recombinant DNA technology has surely made its way to being one of the most

significant technologies in biology, research, medicine, agriculture and industry. The method

is significant because it allows for the synthesis of numerous copies of genes as well as the

insertion of foreign genes into other species to confer new characteristics such as antibiotic

resistance or a different trait. The first time rDNA technology was deployed allowed the mass

production of human proteins on an unprecedented scale at low cost, paving the path for more

detailed research into protein function and medicinal applications.

Back then, treatments to very rare diseases, vaccines, and many others were just things

imaginable but now, with the help of the recombinant DNA technology, paved way in making

advancements for human existence- one of the most significant is the insulin.

This technique offers a wide range of uses and has the potential to improve vital areas

of life, such as health, food security, and resistance to a variety of detrimental environmental

effects.
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References

Griffiths, A. J.F. (2020, October 14). recombinant DNA. Encyclopedia

Pham, P. V. (2018). Medical biotechnology. Omics Technologies and Bio-Engineering, 449-

469. https://doi.org/10.1016/b978-0-12-804659-3.00019-1

Pham, J. V., Yilma, M. A., Feliz, A., Majid, M. T., Maffetone, N., Walker, J. R., Kim, E.,

Cho, H. J., Reynolds, J. M., Song, M. C., Park, S. R., & Yoon, Y. J. (2019). A review

of the microbial production of Bioactive natural products and biologics. Frontiers in

Microbiology, 10. https://doi.org/10.3389/fmicb.2019.01404

Suza, W., & Lee, D. (2021). Genetics, agriculture, and biotechnology. Iowa State University

Digital Press 701 Morrill Rd, Ames, IA 50011.