DNA Cloning

Dr. Keith Dadson

1
What is Cloning?

2
Animal Cloning 101

3
 What is DNA Cloning?
• Processes in molecular biology used to make large amounts of
identical copies of DNA fragments or proteins of interest

1. Isolate gene 3. Put into cloning system

2. Insert into vector

4. Isolate copies (product)

4
 PCR vs DNA Cloning

PCR DNA Cloning

• Performed in a tube • Performed in cells
• Short sequences of DNA • Long DNA sequences
• Rapid multiplication in small volumes • Multiplication in large quantities
• Easy to create recombinant (tagged) • Easy to create recombinant (tagged) gene
gene fragments fragments
• Can be used for investigative purposes • Expression system which allows for mass
• Genotyping production of proteins
• Mutation detection • Therapeutics
• Phylogenetics
• Homo sapiens
• Neadertal
• Denisovan
• Forensics (DNA fingerprinting)
5
 Cloning Applications
• Production of recombinant proteins: • Transgenic organisms:

• Agricultural utility: • Gene Therapy:

6
 Overview

• DNA isolation

• Restriction
Digest

• DNA Ligation

• Transfection 7
 Vectors:
A stable small piece of DNA into which a foreign DNA fragment may be
inserted for cloning purposes. The recombinant vector is then inserted
into an organism without loss of capacity for self-replication
• Plasmid: small circular DNA molecules that can replicate
independently

• Phages: viruses that infect bacteria

• Cosmids: artificial vectors made from combining plasmids

and phages

• Artificial Chromosomes: Yeast (YAC), Bacterial (BAC),

Mammalian (MAC) 8
Different types of vector with their properties.
Vector Basis Size limit of Major application
insert
Plasmid Naturally occurring multi copy ≤ 10 kb Subcloning and gene manipulation, cDNA cloning
plasmids and expression studies.

Phage Bacteriophage λ 10- 20 kb Genomic DNA cloning, cDNA and expression libraries
Cosmid Plasmid containing a 35- 45 kb Genomic library construction.
bacteriophage λ cos site
BACs Escherichia coli F factor plasmid 75- 300 kb Analysis of large genomes.
YACs Saccharomyces cervisiae 100- 3000 kb Analysis of large genomes, YAC transgenic
centromere, telomere, and mice.
autonomously replicating
sequence
MACs Mammalian centromere, 4> 10 mb Still in budding stage for use in animal biotechnology
telomere, and origin of and human gene therapy
replication

9
Bacterial genome: Chromosome & Plasmids
Plasmid

Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000
kilobase pairs.

Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule.

Bacterial Genome: The collection of all of the genes present on the bacteria’s chromosome or its
extrachromosomal genetic elements.

10
Plasmid Conformations

11
Plasmid Conformations

12
Plasmid Maps and Common Elements
• Restriction Sites: e.g. BamHI, EcoRI,
HindIII

• Multiple Cloning Site: site where

insertion will not disrupt replication or markers

• ORI: origin of replication

• Antibiotic Resistance: e.g. ampicillin,

kanamycin, neomycin

•Reporter gene: e.g. luciferase

13
 Overview

• DNA isolation

• Restriction
Digest

• DNA Ligation

• Transfection 14
Restriction Digestion: sticky vs blunt ends
Enzyme cuts at same site on both DNA strands.

Sequence is a Palindrome (read the same forwards and backwards)

Restriction fragments are pieces of DNA created by restriction enzymes.

Restriction fragment can have sticky or blunt ends.

15
 Overview

• DNA isolation

• Restriction
Digest

• DNA Ligation

• Transfection 16
 Ligation
Restriction Digest

Sticky ends:
single-stranded overhangs

Matching overhand base-pair together

Gaps are sealed with DNA ligase
17
 Overview

• DNA isolation

• Restriction
Digest

• DNA Ligation

• Transformation 18
Methods of Genetic Delivery
• Transformation: Gene delivery into bacterial cells

• Transfection: Gene delivery into mammalian cells

• Transduction: Viral delivery of genetic material

19
Bacterial Transformation
Required components:

1. Recombinant Plasmid
2. Competent cells: cells modified to allow passage of foreign DNA

To make cells competent:

• Concentrated calcium salt solution: neutralizes the negative charge

of membrane that allows negatively charged DNA to enter

• Electroporation: strong electrical current draws negatively charged

DNA into cells
20
Mammalian cell transfection
Transfection: The process of introducing nucleic acids into
eukaryotic cells by non-viral methods is defined as “transfection”.
Calcium phosphate DEAE-dextran

1. Chemical Reagents

2. Liposome-mediated
delivery - synthetic
cationic lipids

3. Physical Methods:
electroporation,
sonoporation and
microinjection
synthetic cationic lipids
21
Gene transduction – nucleic acid insertion into the
host genome
1. Most efficient - 90–100% transduction efficiency of
primary cells

2. Engineering a viral vector to carry a gene of interest >

vector is then introduced into a packaging cell line to
produce recombinant viral particles > The particles are
then collected and used to infect the cells of interest.

3. The downside of working with viruses is that they

present biosafety issues.

• Retrovirus Viral Vectors: require mammalian

• Adenovirus host cell 22
Selection and Analysis of Positive Clones
1. Select for transformed bacteria: antibiotic resistance

2. Select for bacteria with recombinant plasmids

• lac selection
• Gene fragment insertion into multiple cloning site
disrupts transcription of β-galactosidase (β-gal)

• β-gal cleaves X-gal (D-lactose mimic) in the growth

substrate to produce blue coloured colonies

• Bacteria with recombinant plasmids will produce no β-gal

and will then appear white 23
Selection and Analysis of Positive Clones
1. Genomic verification

• Agarose gel
• DNA sequencing
• Quantitative PCR
• Southern blot

2. Protein verification

• Western blot
• ELISA
24
RECAP

25
 CRISPR editing & DNA cloning
CRISPR: a method for DNA editing

CRISPR editing requires co-expression of

an endonuclease like Cas9 and a guide
RNA (gRNA) specific to the targeted gene.

CRISPR can be used to:

• Knock out, knock in, or modify a gene
• Induce epigenetic modifications
• Perform Genome-wide screens
• Visualize genomic loci

With respect to DNA cloning:

• An edited gene can be amplified
through cloning
• An edited nucleus can be inserted into
a recipient cell
• Both Cas9 and gRNA can be introduced
via a vector (e.g. plasmid or virus) to
edit the genome in situ
26
 DNA Cloning Applications: Sickle Cell Disease Gene Therapy
Sickle Cell Disease

• Genetic disease caused by mutation of HBB

• HBB: hemoglobin β subunit

• HBB point mutation: glutamic acid → valine

• Stiff sickled red blood cells block blood vessels

N Engl J Med 2021; 384:252-260

CRISPR-Cas9 was used to enhance production of normal/fetal hemoglobin in CD34+ hematopoietic stem and progenitor
cells. The modified cells were delivered to patients with Sickle Cell Disease, increasing the amount of normal
hemoglobin in their blood. 27
 DNA Cloning Applications: Cystic Fibrosis Gene Therapy
Cystic fibrosis

• a genetic disease caused by a CFTR mutation

• CFTR: cystic fibrosis transmembrane conductance regulator

• Mapped by positional cloning in 1985
• 190 kb
• Located on the long arm of chromosome 7 (7q31.2)

• CFTR is embedded in the membrane of epithelial cells

Lancet Respir Med. 2015 Sep;3(9):684-691

Lung delivery of plasmid DNA encoding the CFTR gene was associated with a small but significant benefit
in forced expiratory volume (FEV), a measure of lung performance.
28
Key takeaways
• DNA cloning is used for mass production of identical DNA fragments or proteins of interest

• The delivery system (vector) is chosen based on size of DNA insert and application

• Restriction enzymes used to cut the vector are also used to cut gene of interest to ensure
homologous end joining

• CRISPR/Cas9 can also be used to modify target genes

• Genetic delivery depends on recipient cells (bacterial vs mammalian). Viruses can be used as a
delivery mechanism to either.

• Selection of successful clones is done through antibiotic resistance and reporter genes

• DNA cloning can be used for gene therapy

29
 Thank you
For questions please contact
Professor Sweeney or Professor Jang

30