MIC 210

BASIC MOLECULAR BIOLOGY

LECTURE 4
DNA CLONING

BY
SITI NORAZURA JAMAL (MISS AZURA)
03 006/ 06 483 2132
 Outline
1. Source of DNA
2. Vector
3. Restriction enzyme
4. Ligation
5. Bacteria host
6. Transformation
7. Selection of recombinants
INTRODUCTION TO DNA
CLONING
What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an
original molecule or cell

• To "clone a gene" is to make many copies of it - for

example, by replicating it in a culture of bacteria.
• Cloned gene can be a normal copy of a gene (= “wild
type”).
• Cloned gene can be an altered version of a gene (=
“mutant”).
• Recombinant DNA technology makes manipulating
genes possible.
• To work directly with specific genes, scientists prepare
gene-sized pieces of DNA in identical copies, a process
called DNA cloning
Fig. 20-2 Cell containing gene
Bacterium of interest
1 Gene inserted into
plasmid

Bacterial Plasmid
chromosome
Gene of
Recombinant interest
DNA of
DNA (plasmid) chromosome
2 Plasmid put into
bacterial cell

Recombinant
bacterium

3 Host cell grown in culture

to form a clone of cells
containing the “cloned”
gene of interest

Gene of Protein expressed

Interest by gene of interest
Copies of gene Protein harvested

4 Basic research and

Basic various applications Basic
research research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth
• A preview of gene cloning and some uses of cloned genes
• Most methods for cloning pieces of DNA in the laboratory share
general features, such as the use of bacteria and their plasmids
• Plasmids
 are small circular DNA molecules that replicate separately from the
bacterial chromosome
• Cloned genes are useful for making copies of a particular gene
and producing a protein product
• Gene cloning involves using bacteria to make multiple copies of a
gene
• Foreign DNA is inserted into a plasmid, and the recombinant
plasmid is inserted into a bacterial cell
• Reproduction in the bacterial cell results in cloning of the plasmid
including the foreign DNA
• This results in the production of multiple copies of a single gene
Gene cloning, genetic engineering,
recombinant DNA technology
They‟re more or less the same
It basically means :
joining together DNA from
different sources/organisms,
forming a recombinant DNA
molecule
Then put this recombinant DNA
into a host cell, usually bacteria
The host cell will then replicate
many copies of this recombinant
DNA molecule
Sometimes, we might want to
ask the host cell to use the
genetic information in the
recombinant DNA to make
proteins
Why genetic engineering ?

Medical & health

applications

Production of novel and

important proteins
Insulin.. See chapter 1
Agricultural applications

e.g. GM crops
„golden rice‟ - Inserting the gene
for synthesis of carotene
(Vitamin A) into rice
Cloning genes for scientific studies
Basic of DNA Cloning
The basics of cloning
You need :
1) Source of DNA - to be cloned
2) Choice of vectors – to carry,
maintain and replicate cloned gene in host
cell

3) Restriction enzymes - to cut DNA

4) DNA ligase - to join foreign and
vector DNA  recombinant DNA
5) Host cell – in which the recombinant
DNA can replicate
1) Source of DNA

• Genomic DNA
– DNA extracted from cells and purified

• cDNA
– by reverse transcription of mRNA

• Amplified DNA
– using Polymerase Chain Reaction

• Synthetic DNA
– DNA made artificially using a machine
2) Vector

• to carry the ligated foreign gene into the host cell

• maintain the foreign gene in the host cell

• Replicate

• pass into new cells during cell division

• Expressed the cloned foreign gene to make a

protein
Different types of cloning vectors

•plasmids

•bacteriophage l, M13
•Cosmids, phagemids
•Artificial chromosomes
BAC, YAC, MAC etc.
Plasmid
• Extrachromosomal DNA found in
bacteria & fungi
• Close circular DNA molecules,
supercoiled
• Can replicate autonomously,
independent of chromosome
• Can be transfer to other cells by
conjugation
• Can be integrated into the
chromosome
• In nature, plasmids carry genes that are not essential under normal conditions
• But confers a survival advantage under extreme conditions eg. resistance to
antibiotics, metabolism of unusual substrates
• Number of plasmid per cell - controlled by plasmid itself
High copy number > 100 /cell; low copy number < 20 /cell
• Plasmid incompatibility – the presence of one plasmid in a cell excludes other
plasmids
 pBR322 – a high copy number plasmid

Important DNA elements :

1. The rop (or sometimes ori)
origin of replication, so that the
plasmid can be maintained &
replicated in the host cell
2. Antibiotic resistance marker
genes (ApR for ampicillin
resistance and TcR for
tetracycline) so that we can
select
3. Unique restrcition sites (EcoRI,
PvuI etc) so that we can cut the
plasmid in one place only.
and insert the foreign gene we
want to clone
3) Restriction enzyme

> Type II Restriction endonuclease

• Enzymes found in some microorganisms

• Natural role to destroy invading foreign DNA

– eg. bacteriophage DNA

• Recognizes very specific short sequences of DNA

– Each enzyme has its own recognition sequence/ site
– Sometimes two different enzymes have the same recognition
sites, in which case they are known as isoschizomers

• Cuts DNA in very specific manner

• Technically – one Unit of RE will completely digest 1 ug

of substrate DNA in a 50 ul reaction volume in 60 minutes
Restriction enzymes cut DNA at very specific sequences

• HindIII PstI

• EcoRI FatI

• SexAI SspI

Recognition sites – always palindromic

-Formation of hairpin loops
How REs cut DNA

Sticky ends can re-anneal by base-pairing

Sticky ends has complementary overhangs
- allows for proper reannealing and joining of DNA molecules
Bacterial transformation
Inserting the recombinant DNA molecule into a Competent E.coli cell
The cells must be made competent be treating with CaCl2 or very little
DNA will be taken up.
Selecting for transformants carrying recombinant DNA

No vector or recombinant DNA

– will not grow on media + ampicillin

Vector only
– will grow on media + ampicillin

Recombinant DNA (vector + insert) –

will grow on ampicillin
This is the one we want !

The goal of any cloning experiment is to obtain transformants carrying

cloned insert DNA. There are several strategies to maximise these
The goal of any cloning experiment is to obtain transformants carrying
cloned insert DNA.

There are several strategies to maximise these

1. Directional cloning

Use two different restriction enzymes to cut each end of the vector
(and also the foreign DNA you want to clone)
- Generate different sticky ends – cannot self ligate
EcoRI BamHI
EcoRI BamHI
3. Dephosphorylation of
vector
-both the 3‟OH group and
5‟PO4 group are required for P
P
ligation
-if the 5‟PO4 groups on the
vector ends are removed –
cannot self-ligate
-Using a phosphatase
enzyme
-e.g calf intestinal
phosphatase etc.
Blue white selection – lacZ complementation

The vector contains a portion of the E.coli LacZ gene.

A multiple cloning site (MCS) sequence is inserted into the LacZ‟ fragment
 The LacZ gene codes for the b-galactosidase enzyme

The b-gal enzyme

hydrolyses lactose into
glucose and galactose
The LacZ gene can be broken into two parts, a and b
- each part encoding a fragment of the b-galactosidase enzyme

LacZb’

Inserted into
plasmid vector LacZa

b- fragment
A fully active enzyme can be reconstituted from both fragments
LacZb’

Inserted into
plasmid vector LacZa

b- fragment

The b-gal enzyme can

also hydrolyse a colorless
substance called X-Gal
into glucose and a blue
color pigment
To do blue white selection, the gene of interest is cloned into the MCS

Gene you
want to clone

Transformants are plated onto a medium containing :

o Antibiotic for selection
o IPTG to induce expression of the LacZ’
o X-Gal to detect the presence of b-galactosidase
Transformants with vector only :
o LacZ is expressed  a fragment is produced
o Complements b-fragment to form fully active enzyme
o Hydrolyses X-Gal  Blue color colonies
Transformants with recombinant DNA:
o LacZ is destroyed by insertion of foreign gene  no a fragment
o Cannot form fully active enzyme
o No hydrolysis of X-Gal  White color colonies
Just to remind you the basic steps….
Sometimes, a simple cloning vector is not good enough

We might want to ask the bacteria cell to make proteins using

information on the cloned gene
We need to use an expression vector
Expression vector
- clone foreign gene AND make foreign
protein
- requires extra DNA elements
Promoter – to initiate transcription –
synthesis of mRNA
Terminator – to stop transcription
Fusion tags – for making fusion proteins
e.g. Histidinex6, c-myc, HA, GFP
In frame MCS
Other things – e.g. Poly-A sites
Recombinant Insulin – not as easy as it looks
The insulin molecule as coded by DNA
Active insulin molecule

C-peptide is removed

Disulfide bonds formed between Peptide A & B

Not done by bacterial cell !

Production of recombinant insulin – „Humulin‟ in E.coli

DNA for peptide A and Peptide B – synthesized chemically

Peptide A – 21 amino acids – 63 nucleotides + ATG + stop codon
Peptide B – 30 amino acids – 90nucleotides +ATG +stop codon

Clone into a different plasmid vector s– into the gene for B-galactosidase

Both DNA‟s were cloned in frame with the b-gal gene

Expressed as fusion proteins – Peptide (A or B) + part of b-gal

This is necessary – otherwise the small peptides will be quickly degraded

Fusion with b-gal stabilises the peptides
Expression driven by the LacZ promoter

Fusion proteins are purified from the cells

The B-gal part is then cleaved off by reacting with cyanogen bromide
which cleaves methionine

The peptide and then purified and chemically reacted to form disulfide bonds

What is the problem of this approach ?