1. MIC 210
BASIC MOLECULAR BIOLOGY
LECTURE 4
DNA CLONING
BY
SITI NORAZURA JAMAL (MISS AZURA)
03 006/ 06 483 2132

2. Outline
1.
2.
3.
4.
5.
6.
7.
Source of DNA
Vector
Restriction enzyme
Ligation
Bacteria host
Transformation
Selection of recombinants

3. INTRODUCTION TO DNA
CLONING

4. 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

5. Fig. 20-2
Cell containing gene
of interest
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Gene of
interest
DNA of
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
Interest
Protein expressed
by gene of interest
Copies of gene
Basic
Protein harvested
4 Basic research and
various applications
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth

6. • 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

7. 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

8. Why genetic engineering ?
Medical & health
applications
Production of novel and
important proteins

9. Insulin.. See chapter 1

10. Agricultural applications
e.g. GM crops
„golden rice‟ - Inserting the gene
for synthesis of carotene
(Vitamin A) into rice

11. Cloning genes for scientific studies

12. Basic of DNA Cloning

13. 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

14. 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

15. 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

16. Different types of cloning vectors
•plasmids
•bacteriophage l, M13
•Cosmids, phagemids
•Artificial chromosomes
BAC, YAC, MAC etc.

17. 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

18. 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

19. 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

20. Restriction enzymes cut DNA at very specific sequences
• HindIII
PstI
•
EcoRI
FatI
•
SexAI
SspI
Recognition sites – always palindromic
-Formation of hairpin loops

21. How REs cut DNA
Sticky ends can re-anneal by base-pairing

22. Sticky ends has complementary overhangs
- allows for proper reannealing and joining of DNA molecules

25. Bacterial transformation

27. 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.

28. 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

29. 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

30. 3. Dephosphorylation of
vector
-both the 3‟OH group and
5‟PO4 group are required for
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.
P
P

31. 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

32. The LacZ gene codes for the b-galactosidase enzyme
The b-gal enzyme
hydrolyses lactose into
glucose and galactose

33. 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

34. 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

35. 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

36. 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

37. 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

39. Just to remind you the basic steps….

40. 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

41. 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

42. Recombinant Insulin – not as easy as it looks
The insulin molecule as coded by DNA

44. Active insulin molecule
C-peptide is removed
Disulfide bonds formed between Peptide A & B
Not done by bacterial cell !

45. 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

46. 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 ?