Amplification by Cloning

Molecular Biology Lab/ 1450452 Racha Al-Khoury

 Cloning

Subcloning: Cutting a piece of DNA from one organism and

inserting it into a vector where it can be replicated by a host
organism.

Using nuclear DNA from one organism to create a second

organism with the same nuclear DNA

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 Cloning
Use restriction enzymes to isolate a single gene, place it into a plasmid
vector.

Bacterial cells were then transformed with the recombinant plasmid.

The bacteria host cells replicated the plasmid, producing many copies of
the gene, thus amplifying it.

The practical application was that human protein products, like insulin, which
were used to treat disease, could eventually be produced from recombinant
molecules in the laboratory using bacteria or another host.

Human protein products like insulin could be used in very large quantities
from the recombinant molecule. Patients no longer had to use insulin isolated
from pigs or cows.

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 Restriction Enzymes
They are endonucleases that cut dsDNA or ssDNA at specific
recognition nucleotide sequences known as restriction sites

The first ever restriction enzyme isolated (that recognized

specific sites generating blunt and sticky ends) was back in
1970.

Since then, over 3000 restriction enzymes have been studied,

and more than 600 are currently commercially available and
used in DNA manipulation techniques.
 Nomenclature of Restriction
Enzymes

Each enzyme is named after the bacterium from which it was

isolated using a naming system based on bacterium genus, species,
and strain.

Ex: EcoRI

E: Escherichia (Genus)
co: coli (species)
R: RY13 (strain)
I: First identified (order of identification in the bacterium)
 Types of Restriction Enzymes
They are categorized into three general groups:

Type I, Type II, and Type III

These are grouped based on their:

- Composition (number of subunits)

- Enzyme co-factor requirements
- The nature of their target DNA sequence
- Position of their DNA cleavage site relative to the
target DNA sequence
 How do they work???
Restriction Enzymes recognize a specific sequence of nucleotides, and
produce a double-stranded cut in the DNA to generate either:
What kinds of bonds are broken
when restriction enzymes cut?

phosphodiester bond
 Choice of Restriction Sites/Enzymes
Once you have your gene, you need to design a way to get it into your
plasmid

How???

• You must choose restriction sites that are available in the plasmid
you are cloning into.

• They must not appear in your gene (silent mutation can remove
unwanted sites in your designed gene).

• Restriction sites must exist only once in your plasmid

 Ligation
Ligation is the process of joining two pieces of DNA from
different sources together through the formation of a
covalent bond.

DNA ligase is the enzyme used to catalyze this reaction.

DNA ligation requires ATP.

+
DNA Ligase

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 DNA Ligase

The most widely used DNA ligase is derived from the T4 bacteriophage.

T4 DNA ligase requires ATP as a cofactor. Common name: T4 DNA

ligase (ATP)

A DNA ligase from E. coli is also available, but is not commonly used.
it uses NAD as a cofactor. Common name: DNA ligase (NAD+)
 DNA Ligase Mechanism

The reaction occurs in three stages in all DNA ligases:

• Formation of a covalent enzyme-AMP intermediate linked to a

lysine side-chain in the enzyme.

• Transfer of the AMP nucleotide to the 5’-phosphate of the nicked

DNA strand.

• Attack on the AMP-DNA bond by the 3’-OH of the nicked DNA

sealing the phosphate backbone and resealing AMP. 
DNA Ligase Mechanism
 Bacteriophage T4 DNA ligase
Characteristics

Molecular mass: Is a single polypeptide with a M.W of 68,000 Dalton. 

pH: The maximal activity pH range is 7.5-8.0. 

Ions: The presence of Mg++ ion is required and the optimal concentration is 10mM.

Temperature: The optimal incubation temperature for T4 DNA ligase is 16 °C.  When
very high efficiency ligation is desired (e.g. making libraries) this temperature is highly
recommended.  However, ligase is active at a broad range of temperatures. 

For routine purposes such as subcloning, convenience often dictates incubating time
and temperature-ligations performed at 4C overnight or at room temperature for 30
minutes to a couple of hours usually work well.
 Plasmid Vectors
Plasmids:

- Are circular pieces of DNA found naturally in bacteria.

- Can carry antibiotic resistance genes, genes for receptors, toxins or

other proteins.

- Replicate separately from the genome of the organism.

- Can be engineered to be useful cloning vectors.

- Can be designed with a variety of features:

1- Antibiotic resistance
2- Colorimetric “markers”
3- Strong or weak promoters for driving expression of a protein
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 Cloning vectors
Antibiotic Resistant
Gene

Multiple Cloning
Site

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 Cloning a Piece of DNA
AvaI AvaI

AvaI
" 5´ 3´

Cut plasmid vector Excise DNA insert of interest

with AvaI from source using Ava I

Ligate the insert of interest

into the cut plasmid

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 Restriction Digestion Reaction
You will need to set up a restriction digest of your plasmid vector and your DNA of
interest.

Restriction enzymes all have specific conditions under which they work best. Some
of the conditions that must be considered when performing restriction digest are:
temperature and the purity of the DNA.

Purify your DNA fragment:

The insert of interest that you want to clone into your plasmid needs to be separated
from the other DNA

You can separate your fragment using Gel Electrophoresis

You can purify the DNA from the gel by cutting the band out of the gel and then
using a variety of techniques to separate the DNA from the gel matrix

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 Today’s Experiment

You are handed two samples:

1- pUC19 (2686bp)
2- Mitochondrial gene flanked with BamHI and HindIII by PCR (257 bp)

Multiple
cloning
site

Objective:
Clone the mitochondrial gene into the
pUC19 multiple cloning site using
BamHI and HindIII restriction enzymes
and then check the final construct
expression in competent E-coli cells
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 Today’s Experiment
Amplified Mito-gene

S2
S1
(257bp) product

ne
ne

ge
ge

to -
bp

to-
100

Mi
Mi
300
200
100

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 Restriction Enzyme Digestion Reaction

Separate digestion reactions should be prepared for both pUC19 and the
amplified mitochondrial gene:

In two separate sterile microcentrifuge tubes, mix the following components:

Incubated at 37°C in a water shaker for 1 hour.

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 Ligation reaction

1- In one sterile microcentrifuge tube, add 3ul of digested pUC19 and 9ul of
digested Mitochondrial DNA.
2- Then add 4ul of 10X T4 DNA ligase buffer followed by 3ul of distilled water.
3- Finally, bring the T4 DNA ligase on ice to your bench and add 1ul of the T4 DNA ligase.
(T4 DNA Ligase should be added last).
4- Cap the tube properly and incubate in a 16°C water bath for half an hour.
5- Remove your sample from the 16° water bath and chill on ice.

Store your sample at -20°C for the following week

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To be followed…

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