RECOMBINANT DNA

Horace Juberry Chikagwa

[email protected]
27 Oct 185 – 6 Jan 1919

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 Recombinant DNA Technology
• Production of a unique DNA mlcl by joining together two or more DNA fragments not formally
associated with each other.
• Genetic engineering is lab-based technology that changes DNA make up of an organism.

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 Recombinant DNA
A B C D E F G H I J K L M N O P Q R ST UVW XY Z
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Breakage & joining of DNA strands

A B C D E F G h I j k l m O P Q R ST UVW XY Z
Abcd ef g HIJKLMNo p q rst uv w xyz
Best studied in yeast, bacteria and phages

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 Recombinant DNA
• Recombinant DNA (rDNA) is a form of artificial DNA
made thr the combi or insertion of one or more DNA
strands hence combining DNA sequences within different
spp.
rDNA mlcl can enter a cell and
replicate autonomously or after
it has been integrated into a
chromosome.

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Recombinant DNA Technology

Recall

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 Recombinant DNA
• In vivo, cells often undergo recombination.

• Eukaryotic Meiosis – cross over.

• To produce rDNA, one must cut precise sites of

vector and insert the DNA of interest.

• Why? To produce more of the gene of interest or

its products.

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 Recombinant DNA
Has allowed molecular Bio
to come to full circle

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 rDNA Tech Applications

1. Molecular basis of diseases

• Understanding molecular basis
of:
• Familial
hypercholesterolemia
• Sickle cell disease
• Thalassemias
• Cystic fibrosis
• Muscular dystrophy

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2. Production of proteins
Protein Use
Human insulin Treatment of diabetes
Human growth hormone Treatment of some endocrine disorders like
dwarfism
EPO To treat anemia
Interferons Resistance to viral infection
Coagulation factors IX & X Treatment of blood-clotting disorders
(hemophilia)
Tissue-type plasminogen activator Blood clot lysis after heart attack & stroke
Bovine growth hormone Milk production in cows
Interleukins Wound healing, cancer, immune deficiencies
Superoxide dismutase Used during surgery

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 rDNA Tech Applications
3. Gene Therapy

• Is introduction of normal genes into individuals

with defective genes.

• Gene therapy for sickle cell disease, thalassemia,

adenosine deaminase deficiency may be devised.

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 rDNA Tech Applications
4. Transgenic Animals
• Transgenic = containing genetic material into which
DNA from a different org has been artificially added.
• Genes introduced into fertilized eggs from which
transgenic animals develop.
• These transgenic animals produce normal offspring.
• To improve animal products for human nutrition &
decrease livestock diseases.

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 rDNA Tech Applications
5. Commercial Applications
• Agricultural – plants that are
efficient at fixing nitrogen, resistant
to diseases, pests, herbicides,
drought & temp extremes.
• Industrial – production of enzymes
used in detergents, sugar and
cheese.

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 rDNA Tech - Background
1972: Paul Berg successfully inserted bacterial DNA into viral DNA in his studies
on tumor virus SV40.
The first DNA mlcl with segments from different organisms was thus made.

Called Hybrid DNA or recombinant DNA.

It was a gateway to production of bacteria that produce

substances used in medicine. The Father of Genetic
Engineering
(American Biochemist 30 Jun 1926 –
15 Feb 2023)

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 rDNA Tech - Background
1973: Helbert Boyer (Uni of California) & Stanley Cohen (Stanford Uni) reported
orgs that combined and replicated rDNA.
They demonstrated impact of DNA recombinant engineering on Medicine,
Pharmacology, Industry & Agri.
They used:
• Living orgs able to serve as carriers of genes from another
org.
• Enzymes to cleave and rejoin DNA fragments that contain
such genes.
• DNA mlcl from one org targeted & manipulated to insert
into DNA of another org.

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 rDNA Tech:
Basic Principle

1. Gene of interest isolated and inserted into vector

(plasmid, phage, viral genome).

2. Plasmid introduced back into host cell

3. Host cell cultured to make more copies

of gene of interest hence protein

4. Harvested protein used in basic research

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 rDNA Tech - Steps
A. Identification & isolation of the gene of interest.
• Obtained from:
i. Genomic lib.
ii. cDNA lib.
iii. Chemically synthesizing (if sequence is known).
iv. Amplification of number of copies by PCR.

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rDNA Tech - Steps

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 rDNA Tech - Steps
B. Insertion of gene of interest in a suitable vector.
• Vector = a DNA mlcl that can replicate inside a
host to which a gene of interest is integrated for
cloning.
• E.g.
• Plasmids, bacteriophages, cosmids, BAC, yeast
vectors, shuttle vectors, etc.

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 rDNA Tech - Steps
Plasmid isolated from E. coli

Restriction enzyme used to

cut plasmid at restriction sites

Gene of interest is
incorporated into the plasmid

Cutting ends sealed by ligase

Chimeric / rDNA is produced

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 rDNA Tech - Steps
C. Introduction of vector into a suitable host
• Methods to transfer vector into a host include:
i. Physical – electroporation, microinjection, liposome
mediated gene transfer, silicon carbide fibre mediated,
ultrasound mediated, DNA transfer via pollen.
ii. Chemical – PolyEthelene Glycol (PEG) mediated, CaCl
mediated, DEAE dextran mediated.
iii. Transformation – DNA imbibition by cells, tissues or
organs.

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 rDNA Tech - Steps

Transformation

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 rDNA Tech - Steps
D. Selection of the transformed host cell
• Three types of bacterial cells are yielded after the previous
step.
• Non-transformed – without any change.
• Transformed cells – with unaltered vector.
• Transformed cells – with the recombinant vector (to be isolated).
• Cells with recombinant vector can be selected by
antibiotic resistance in selective medium, looking for
visible characters, adding substrate for coloration or
blotting test.

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 rDNA Tech - Steps

Non-transformed Transformed cell with Transformed cell with

bacterial cell unaltered vector recombinant vector

Many colonies belong to these classes Cells to be isolated

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 rDNA Tech - Steps
E. Multiplication or expression of the introduced
gene in the host.
• Aim is to either make:
i. more copies of the gene of interest or
ii. gene products i.e. protein from the gene within
the host cell.

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Tools of rDNA Tech

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 Restriction Enzymes
• Naturally produced by bacteria.
• Degrade DNA mlcls by breaking the phosphodiester bonds
that link one nucleotide to the next in a DNA strand.
• They are categorized into:
• Exonucleases – remove terminal nucleotides of DNA mlcl
by breaking the phosphodiester bond.
• Endonucleases – break internal phosphodiester bonds.

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 Exonucleases

Remove nucleotides, one at a time from the end

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 Restriction Endonucleases
• Aka Molecular scissors.
• They fragment viral DNA as soon as it enters
bacterial cell.
• They recognize specific nucleotide sequences
in a DNA strand called restriction sites.
• They then bind to the sequence and cleave the
DNA at a particular site within the recognition
sequence.

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 Restriction Endonucleases
• There are three types of REs:
1. Type I Restriction Endonucleases.
2. Type II Restriction Endonucleases.
3. Type III Restriction Endonucleases.
• Most Type II REs recognize hexanucleotide (6 bp)
sequence.
• Some recognize 4 or 5 bps.
• Recognition sites have palindromic nucleotide sequence
(read similar backward and forward)

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Type II Restriction Endonuclease Characteristic Properties

• Most commonly found and highly useful in rDNA

research.
• Each restriction site of Type II endonucleases is a
palindrome (reads the same forward and backward
on opp strands of DNA).
• Highly specific cleavage
of DNA strands.

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Type II Restriction Endonuclease Characteristic Properties

• Restriction sites are usually:

• 4 base pairs – Bfal, Alul, Hpall
• 6 base pairs – EcoRI, BamHI, PvuII
• 8 base pairs – NotI, Pmel, SbfI
• Frequency of restriction sequences in a DNA mlcl.
• 4n where n = number of bases in a particular
restriction site.
• For example if n = 6, then freq = 7776 nucleotides.

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THANK YOU

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