Introduction to Bioengineering
Manisa Celal Bayar University
Department of Bioengineering
2023-2024 Fall Semester
Hilal Betül KAYA AKKALE, PhD
[email protected] Recombinant DNA Technology
Recombinant DNA technology is the manipulation
and combination of DNA molecules from different
sources.
An Overview of Recombinant DNA Technologies
1. Gene of interest (DNA) is isolated
(DNA fragment)
2. A desired gene is inserted into a
DNA molecule - vector
(plasmid, bacteriophage or a viral genome)
3. The vector inserts the DNA into a
new cell, which is grown to form a
clone.
(bacteria, yeast, plant or animal cell)
4. Large quantities of the gene product
can be harvested from the clone.
Tools for Genetic engineering
1. Restriction Enzymes
• Naturally produced by bacteria – restriction endonucleases
• Natural function - destroy bacteriophage DNA in bacterial cells
• Cannot digest host DNA with methylated C (cytosine)
• A restriction enzyme
• Substrate –DNA -recognizes one particular nucleotide sequence in DNA
and cuts the DNA molecule (breaks down the bond between two
nucleotides)
sticky ends blunt ends
Selected Restriction Enzymes Used in
Recombinant DNA Technology
Tools for Genetic engineering
2. Ligase
• DNA ligase is an enzyme that can link together DNA strands that
have double-strand breaks (a break in both complementary strands
of DNA).
• DNA ligase has extensive use in molecular biology laboratories for
genetic recombination experiments
Tools for Genetic engineering
3. Vectors
• Vectors - small pieces of DNA used for cloning (the gene to be
inserted into the genetically modified organism must be combined with
other genetic elements in order for it to work properly)
• Requirements of the Vector
1. Self-replication - able to replicate in the host (origin of
repliction)
2. Cloning site (site for recognition of restriction nucleases)
3. Promoter (and operator) - to support the gene (new DNA) expression in the
host
4. Selectable marker – antibiotic resistance
5. Proper size
Hosts for DNA Recombinant Technology
1. Bacteria
- E. coli is used because it is easily grown and its
genomics are well understood.
• Gene product is purified from host cells
2. Yeasts - Saccharomyces cerevisiae
• is used because it is easily grown and its genomics are known
• may express eukaryotic genes easily
• can continuously secrete the gene product.
• can be easily collected and purified
3. Plant cells and whole plants
• May express eukaryotic genes easily
• Plants are easily grown
4. Mammalian cells
• May express eukaryotic genes easily
• can be used specifically in medical application
• Harder to grow
Insert the naked DNA into a host cell
Transformation is the process by which foreign genes or DNA fragments are
introduced into a host organism,
Indirect methods are used for plant
transformation.
Recombinant DNA technology - Cloning
A process of producing genetically modified organisms
A multi-step process.
1. Isolating and copying the genetic material of interest (DNA fragment ).
2. Building a construct (recombinant DNA - vector and desired gene)
containing all the genetic elements for correct expression.
3. Inserting the vector into the host organism, directly through injection or
transformation.
4. Selecting the cells expressing that gene by growing under positive selection
(of an antibiotic or chemical) – clone .
5. Growing successfully the clone (transformed organisms).
Illustration of various applications of Recombinant
DNA Technology
