CLONING

 Objectives of the lesson:

At the end of this lesson students will be able to

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1. Recall the importance of genetic recombination

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2. Recognize the basic steps in cloning

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3. List down types of cloning

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CLONING

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 Cloning is the process of producing a strain of DNA and
then inserting that DNA into a host where it will
replicate.

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 The replicated DNA is called a clone.

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 Cloning as a technique has many uses.

 For example, it can be used to replicate rare hormones and

proteins such as insulin and interferon that have much medical usage.
 Recently
2 cloning has been taken to far a far greater extent.

Whole organisms have been reproduced from DNA taken from

other bodies
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STEPS IN CLONING
CUTTING DNA
 Restriction endonucleases
(restriction enzymes)
 sticky ends
 blunt ends

 Nomenclature
 EcoRI
 E = genus (Escherichia)
 co = species (coli)
 R = strain
 I = # of enzyme
Blunt & Sticky ends
PASTING DNA

 Complementary ends
(sticky ends) H-bond

 Ligase forms
phosphodiester bond
to seal strands
together.
 A PLASMID VECTOR FOR CLONING

1. Contains an origin of replication, allowing for

replication independent of host’s genome.

2. Contains Selective markers: Selection of cells

containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)

4. Easy to be isolated from the host cell.

Plasmid vectors
EXPRESSION VECTOR
HOW TO CLONE DNA

 Isolation of cloning vector

(bacterial plasmid) & gene-
source DNA (gene of interest)
 Insertion of gene-source DNA
into the cloning vector using
the same restriction enzyme;
bind the fragmented DNA with
DNA ligase
 Introduction of cloning vector
into cells (transformation by
bacterial cells)
 Cloning of cells (and foreign
genes)
 Identification of cell clones
carrying the gene of interest
HOW TO CLONE A GENE

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 Restriction Digests

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 Vectors

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 Gene of Interest

 Putting them together

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RESTRICTION ENZYMES
USES OF RESTRICTION ENZYMES

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 Cloning

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 Chromosome or plasmid mapping

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 Epidemiology

 Genetic analysis

 Southern blotting

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 Probe production
Digest
RESTRICTION ENZYMES

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TTGCAA

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AACGTT

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Digest
SINGLE ENZYME DIGEST

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Up to 1 g of DNA  X L
X units of Enzyme#  1 L

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10X buffer  1 L

Water*  Up to 7 L

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___________________ ___________________
10 L reaction mix  10 L reaction mix

*(mol bio grade)

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Vector
VECTORS
Multiple Cloning Site

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Reporter

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GOI

Selection ORI
Marker 16
Digest

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In molecular biology, a reporter gene (often
simply reporter) is a gene that researchers attach to a
regulatory sequence of another gene of interest in cell

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culture, animals or plants.

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Certain genes are chosen as reporters because the
characteristics they confer on organisms expressing
them are easily identified and measured, or because
they are selected markers.

Reporter genes are often used as an indication of

whether a certain gene has been taken up by or
expressed in the cell or organism population. 17
ANTIBIOTIC SELECTION
(TRANSFORMATION)
 A resistance gene in the plasmid allows only those bacteria that

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have taken up the plasmid to grow in the media (ie. those
bacteria that are transformed)

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 Ampicillin

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 Kanamycin
Only two of several
that can be used in
cloning experiments

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Scharf Stock Photos

TRANSFORMATION
SCREENING OF THE CLONE

 The medium in this petri

dish contains the antibiotic
Kanamycin

 The bacteria on the right

contain Kanr, a plasmid
that is resistant to
Kanamycin, while the one
on the left has no resistance

 Note the difference in

growth
BLUE/WHITE COLOR SCREENING

lacZ lacZ insert

functional enzyme nonfunctional enzyme

X-gal product X-gal product
SELECTING COLONIES WITH
RECOMBINANT PLASMIDS
Vector
ORI

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 Origin of Replication
 Place where DNA Polymerase starts replication; in both
directions

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 Determine the number of copies / cell

 For cloning –
 You can only get one kind of vector into a bacteria
 unless you use two different antibiotic selection
markers

 unless you use two different ORIs 23

SINGLE ENZYME DIGEST
AND LIGATION - GOI

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 Blunt: PCR product as is

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Nega B
 Sticky End Ligation:

 Must cut PCR product before ligation

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SINGLE ENZYME DIGEST
AND LIGATION - VECTOR

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 Blunt Cut

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 Sticky End Cut
 5’ overhang

 3’ overhang

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SINGLE ENZYME DIGEST
AND LIGATION - VECTOR

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 Blunt End Ligation

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 Sticky End Ligation

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USES OF
SINGLE DIGEST CLONING

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 Cloning of PCR products for sequencing

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SINGLE ENZYME DIGEST
AND LIGATION - VECTOR

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• Blunt End Ligation

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• Sticky End Ligation

?
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Vector
VECTORS
MCS

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Reporter

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GOI

Selection ORI
Marker 29
DIRECTIONAL CLONING
FOR PROTEIN EXPRESSION

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 In vector
 Promoter,
RBS, ATG of Reporter gene

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 Clone GOI before reporter

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ATG of GOI ATG of Reporter

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 CLONING
Cut DNA and plasmid with same
restriction enzymes HindIII & BamHI

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HindIII & BamHI

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GOI
Plasmid/Vector

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Gene Of Interest in DNA

AmpR
GOI

Ligate the plasmid and POI

DNA fragment together POI

POI 31

AmpR Protein Of Interest production

DIRECTIONAL CLONING
FOR FUSION PROTEIN EXPRESSION

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 In vector
 Promoter, RBS

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 Clone GOI with ATG before reporter/tag
 Flexible spacer before reporter/tag

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ATG of GOI Fusion with Reporter

 MUST BE IN FRAME with tag

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 FUSION PROTEIN CLONING
Cut DNA and plasmid with same
restriction enzymes HindIII & BamHI

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HindIII & BamHI
GFP

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GOI
Plasmid/Vector

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Gene Of Interest in DNA

AmpR

GOI GFP
POI GFP
Ligate the plasmid and
DNA fragment together POI GFP

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GFP

AmpR Protein Of Interest production

DIRECTIONAL CLONING

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 Do double digest of vector
 Gel purify

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 Do double digest of PCR product

 Ligate

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GEL PURIFICATION

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 Run digested DNA on agarose gel
 Cut desired DNA out of gel

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 Purify DNA from gel matrix

 Proceed with modifications of vector

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 Proceed with ligation

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DIGESTION & GEL PURIFICATION
1 2 3 4 5 6

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Lane
1. Uncut

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2. single cut

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3. double cut

4. PCR cut

5. ligated uncut

6. ligated cut

(this is the QC
or screening result) 36
VECTOR MODIFICATIONS

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 Blunt Cut

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P

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 Sticky End Cut

P
P
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Remove phosphate groups with Alkaline Phosphatase
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POLYMERASES
CLONING PCR PRODUCTS

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 Blunt Cut

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 Must use blunt DNA polymerase

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 Pfu for example (also great for accuracy)
 Must phosphatase treat vector
 Yield is usually pretty low
 Lots of new ligases & buffers to help

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CLONING PCR PRODUCTS

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 TA Cloning

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A
A

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 Taq makes 3’A overhangs
 Cut vector with blunt cutter

T
T
 T-tailthe vector with Taq and dTTP (72°C)
 Ligate
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CLONING PCR PRODUCTS

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 TA Cloning

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T7 or M13F T A
A T T3 or M13R
 Sequencing from sequencing sites in vector

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MUTAGENESIS

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 Insertion or deletion at RE site
 Random mutagenesis with nasty chemicals like BrdU

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then screen
 Site-directed mutagenesis permits single base changes

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 Kits
available
 More than one strategy

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Mutation Nobel Moment
Dr. Michael Smith (1932-2000) well known

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scientist from British Columbia, Canada
 He won the Nobel Prize in 1993 for a technique

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that allows any specific DNA sequence at a
particular site in a gene to be mutated

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 This allows us to create and study mutant
proteins

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One strategy for SDM

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 PCR #1

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Product Product

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One strategy for SDM

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• PCR #2

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Product

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One strategy for SDM
RECAP

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PCR cloning 1 Expression cloning
Blunt cut vector Double digest vector

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Phosphatase treat Phosphatase treat
PCR with no overhang
PCR primers

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Ligate
Double digest PCR
PCR cloning 2
product
Blunt cut and T-tail
Ligate
vector
Mutagenesis
PCR with A overhang
Ligate
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CARE AND FEEDING
OF RESTRICTION ENZYMES

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 Centrifuge briefly upon receipt
 Store and keep at -20°C

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 Do not hold bottom of tube

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 Do not pipette to the bottom of the tube

 Do be aware of glycerol effects

 Pipetting
 Inhibition

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SINGLE ENZYME DIGEST

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Up to 1 g of DNA  X L
X units of Enzyme#  1 L

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10X buffer  1 L

Water*  Up to 7 L

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___________________ ___________________
10 L reaction mix  10 L reaction mix

*(mol bio grade)

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DOUBLE ENZYME DIGEST

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 Up to 1 g of DNA  X L
 X units of Enzyme 1  1 L ^

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 X units of Enzyme 2  1 L ^

 10X buffer*  1 L

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 Water  Up to 6 L

___________________ ___________________
 10 L reaction mix  10 L reaction mix

^Must consider max

*(Selection of buffer) glycerol at 5% of total
volume
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DOUBLE ENZYME DIGEST

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 Up to 1 g of DNA  X L
 X units of Enzyme 1  1 L

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 X units of Enzyme 2  1 L

 10X buffer*  2 L

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 Water  Up to 15 L

___________________ ___________________
 20 L reaction mix  20 L reaction mix

Must consider max

*(Selection of buffer) glycerol at 5% of total
volume
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REBASE

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www.rebase.
 Search for restriction enzyme digest sequences

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 Select commercial or special enzymes

 Find isoschizomers, methylation sites, enzymes that

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generate compatible ends
 Star activity

 Generate a gel pix of your sequence

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GENE CONSTRUCTION KIT
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Ligate
LIGATION

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100ng vector DNA  5•LX L
33ng insert DNA  5 L
• X L

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5X Ligation Buffer  4 μl
• 4 μl
T4 DNA Ligase* (3U/μl)  1 μl
• 1 μl

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Nuclease-Free Water  5 L
• X L
___________________ ___________________
___________________
 20 L reaction mix
20 L reaction mix • 20 L reaction mix

*Newer ligases and buffers mean we can

incubate for 15 min at RT 56
Ligate
DNA LIGASE

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 ATP required as co-factor and co-substrate
 Optimal temperature is below room temperature

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 High concentrations used for faster ligations

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Ligate
LIGATION- ISSUES

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 Molar ratio of plasmid to insert is 1:3 but can try other
variations

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 Old ligation buffer

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Transform
ANTIBIOTIC SELECTION
Filter sterilize

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Add to cooled media
Store stock antibiotics at -20oC
 Ampicillin *subject to breakthrough colonies

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 Kanamycin
 Carbenicillin

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 Tetracycline

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Scharf Stock Photos

Transform

COMPETENT CELLS
 Competent cells made by replacing Mg2+ with Ca2+ or

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Rb2+
 1 in 10,000 cells becomes competent

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 Will incorporate only 1 kind of plasmid (except if

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different ORIs or different antibiotic seletion marker)
 Methylation negative (dam-)

 Endonuclease negative (endA-)

 Restriction modification system negative (hsdR-)

 Recombination negative (recA-)

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Transform

TRANSFORMATION

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Ligated vector  1 L
Competent E.coli  50 L

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Transform TRANSFORMATION OF PLASMIDS
INTO BACTERIA

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Foreign plasmid
with ampR gene

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ampS E. coli cells are prepared in ice-cold Plasmids carrying the ampR gene
CaCl2 solution and kept on ice Enter the E. coli cells

Ampicillin-sensitive (ampS)
E. coli cell

Only ampR E. coli cells 62

Transformed (ampR) E. coli cell grow on agar containing ampicillin

http://www.phschool.com/science/biology_place/labbench/lab6/test1.html
THE LAC OPERON transacetylase

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Lac I O Lac Z Lac Y Lac A

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Lac I bGal

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Permease

Lactose
(Galactose (b14)Glucose)

1. When Glucose is present and Lactose is not

H
2. When Lactose is present and Glucose is not
Glucose
3. Lac I is “leaky”, therefore some low level expression will63
occur so that the organism can test if lactose is present or
not
THE LAC OPERON REPORTER

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Lac I O Lac Z

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Lac I bGal

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H

Glucose
IPTG 64
(Isopropyl
Thiogalactoside)
BLUE/WHITE SCREENING BASED ON LAC
OPERON

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SO WHAT DOES IT MEAN?
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SO WHAT DOES IT MEAN?
 CLONING…
INSULIN

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 Insulin is a protein hormone produced in the pancreas that the
body uses to regulate blood sugar concentrations.

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 Diabetics have lost the ability to produce insulin and must have an
outside source of it.

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In the 1920s, insulin from cows and pigs was isolated and made
available to humans with diabetes. (Though it is not identical to
human insulin.)
 Supply was a major concern since the number of diabetics
was on the rise.
 Cloning
68 insulin became an ideal usage for recombinant
DNA technology.
THE MANUFACTURE OF INSULIN BY CLONING

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 In 1978, Herbert Boyer and colleagues at the University
of California in San Francisco created a synthetic

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version of human insulin using recombinant DNA
technology.

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 The DNA sequence representing the instructions on
growing insulin was separated and then inserted
into the bacterium E. coli.
 The E. coli then produced prodigious amounts of human
insulin
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THE MANUFACTURE OF INSULIN BY CLONING

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CLONING WHOLE ANIMALS

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 In 1997, the sheep “Dolly” was cloned
from an adult sheep.

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Dolly is an exact replica of its “mother”
– the animal from which the cell was

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taken

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CLONING WHOLE ANIMALS

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

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STEM CELLS

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 Most cells in the body of an adult animal
are specialized cells, which have the

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capacity only to reproduce themselves.
 Cells that have the ability to divide and

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give rise to different kinds of specialized
cells are called stem cells

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STEM CELLS

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 At conception, the fertilized egg is a stem cell capable
of dividing and becoming every different kind of cell in

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the adult body.
 They are “Totipotent.”

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In humans, the cells that are produced in the first four
days or so after conception are all totipotent stems.
 At later embryonic stages and even in the grown adult,
there are stem cells with limited potential to grow into
different
74 kinds of cells. These are called “Pluripotent.”
STEM CELLS, 2

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 The medical potential of stem cells, both the totipotent and
pluripotent is enormous.

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If stem cells can be isolated, cultured, and then grafted into
patients, many degenerative diseases could possibly be

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reversed.
 Cells generated from a patient’s own stem cells, for example,
would not be rejected by the body the way that the cells of
donor organs often are.
Stem cells could be used to regenerate brain and nerve cells,
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possibly heart muscle, and many other possible uses.
ETHICAL ISSUES IN MOLECULAR BIOLOGY

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There are ethical issues all the way along in biotechnology because

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human beings are capable of manipulating life as never before.
 Stem cell research raises the issue of where life begins and whether

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cells from a human embryo should be used for another human’s
benefit.
 Present stem cell work concentrates on making regenerative cells for
the cure of diseases.
 But the possibility of cloning whole human beings has to be
considered.
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 Dolly was cloned from a stem cell