Recombinant DNA Technology

Course: Research Techniques

April 6, 2024
Week 10

Gulnaz Khan
Department of Biomedical & Biological Sciences
Learning
objectives
 Recombinant DNA technology basics

 Process of recombinant DNA (rDNA) preparation

 Process of rDNA transformation and screening

 Types of cloning vectors

 Applications of recombinant DNA technology

 Recombinant DNA Technology
Recombinant DNA technology involves the joining of DNA from different species and
subsequently inserting the hybrid DNA into a host cell for its replication, host often a
bacterium.
Recombinant DNA refers to the creation of new combinations of DNA segments that
are not found together in nature. The process is so called “Genetic engineering”
means making change or manipulation in DNA.

The technique was invented by Stanley Cohen and Herbert Boyer in 1973.

Herbert Boyer Stanley Cohen

Recombinant DNA
How to prepare a Recombinant DNA?
Isolate human DNA Isolate Plasmid from Bacterium

Cut with restriction enzymes Cut with restriction enzymes

Ligate human DNA into plasmid DNA of bacteria (cloning vector)

Transform recombinant DNA molecule into host cell (bacterium)

Each transformed cell will divide many times to form a colony of millions of
cells, each of which carries the recombinant DNA molecule (DNA clone)
 Recombinant DNA
Preparation: Process
Recombinant DNA Preparation: Process
Recombinant DNA Preparation: Steps of the Process

 Isolation of DNA

Pure DNA and plasmid is required that is free from RNA, proteins, lipids etc.

 Cutting DNA with restriction enzyme

Staggered cut is required to generate “sticky or over hanging ends” so that

DNA binds with vector

Recombinant DNA Preparation: Steps of the Process

 Joining DNA

Once you have isolated and cut the donor and vector DNAs, they must be joined

together. The DNAs are mixed together in a tube. If both have been cut with the

same restriction enzyme, the ends will match up because they are sticky. DNA

ligase is the glue of molecular genetics that holds the ends of the DNAs together.

DNA ligase creates a phosphodiester bond between two DNA ends.

Recombinant DNA Preparation Steps:
Joining the DNA

Recombinant DNA (rDNA)

Recombinant DNA Preparation: Steps of the Process
 Transform recombinant DNA molecule into host cell

To produce large amounts of the recombinant DNA molecule, it must be

amplified. This is accomplished by transforming the recombinant DNA into a

bacterial host strain.

Cells are treated with CaCl2

rDNA is added

Cells are heat shocked at 42◦C

DNA goes into cell

 Types of a Vector w.r.t function
 Cloning vectors
 Expression vectors
 Cloning Vector: Characteristics
 It must contain origin of replication

 It must be small in size, making it easy to manipulate

 It must be self-replicating inside host cell

 It must possess restriction site for restriction endonuclease enzymes

 Insertion of donor DNA fragment must not interfere with replication property

of the vector

 It must possess some selectable marker gene such that it can be used for later

identification of recombinant cell

 It must possess multiple cloning site

Cloning Vector
 Expression Vector: Characteristics
 It must contain origin of replication

 It must be small in size, making it easy to manipulate

 It must be self-replicating inside host cell

 It must possess restriction site for restriction endonuclease enzymes

 Insertion of donor DNA fragment must not interfere with replication property of the vector

 It must possess some selectable marker gene such that it can be used for later identification of

recombinant cell

 It must possess multiple cloning site

 Transcriptional promotor site

 Transcriptional terminator site

Expression Vector
 Types of Vectors
 Plasmids

 Bacteriophage

 Bacterial artificial chromosomes (BACs)

 Yeast artificial chromosomes (YACs)

 Human artificial chromosomes (HACs)

Vectors that are able to carry larger gene sequences than any other vector

are referred to as artificial chromosomes.

 Types of Vectors
 Bacterial artificial chromosomes (BACs): A bacterial artificial chromosome (BAC)

is an engineered DNA molecule used to clone DNA sequences in bacterial cells

(for example, E. coli). Segments of an organism's DNA, ranging from 100,000 to

about 300,000 base pairs, can be inserted into BACs.

 Yeast artificial chromosomes (YACs): Yeast artificial chromosome (YAC) is a

human-engineered DNA molecule used to clone DNA sequences in yeast cells.

Segments of an organism's DNA, up to one million base pairs in length, can be

inserted into YACs.

 Screening of Transformed Cells

Blue white Screening

It is the most widely used molecular biology technique for the screening of

transformants. This technique uses the activity of B-galactosidase the enzyme coded

by LacZ gene. Naturally B-galactosidase metabolizes lactose to glucose & galactose.

B-galactosidase can also hydrolyze X-gal to form blue color.

X-Gal is a widely used chromogenic substrate for β-galactosidase. It is the analog of

lactose.
Screening of Transformed Cells
Blue white Screening: Screening of
Transformed Cells
 Applications of Recombinant DNA Technology

 Production of recombinant proteins: Recombinant DNA technology is used to

produce large quantities of medicinally important proteins, such as insulin and

growth hormone.

 Gene therapy: Recombinant DNA technology is used to introduce new genetic

material into cells, in order to correct genetic disorders or to treat certain

diseases.

 Agriculture: Recombinant DNA technology is used to create genetically

modified crops that are resistant to pests and diseases, or have improved

nutritional content.
 Applications of Recombinant DNA Technology

 Bioremediation: Recombinant DNA technology is used to create

microorganisms that can break down pollutants and other toxic substances.

 Vaccine development: Recombinant DNA technology is used to produce

vaccines by introducing viral genes into host cells to produce the necessary

proteins for immunity.

 Reference/Reading
Material
 Molecular Cloning: A Laboratory Manual by Joseph Sambrook, David Russell, and Michael W. E. J. (2012)

 Biotechnology: Recombinant DNA Technology" by Satyanarayana, U. and Chakrapani, U. (2016)

 Recombinant DNA: Genes and Genomes - A Short Course" by James D. Watson, Richard M. Myers, Amy A.
Caudy, and Jan Witkowski (2007)

 Principles of Gene Manipulation and Genomics" by Sandy B. Primrose and Richard M. Twyman (2019)

 Gene Cloning and DNA Analysis by T.A Brown