1. RECOMBINANT
DNA
TECHNOLOGY
T H Y

2. What is Genetic
Engineering?
• Genetic engineering ( ) is the transfer of
g g (GE)
genes from one organism to another
through means that do not occur in
nature, but through human intervention.
atu e, t oug u a te e t o
This involves isolating and then moving
genes within and without different species
by recombinant DNA techniques and other
manipulation of the genetic construct
outside the traditional practices such as
sexual and asexual breeding
breeding,
hybridization, fermentation, in-vitro
fertilization and tissue culture.

3. The Term of Gene
Manipulation
Gene Manipulation is d f
l defined as the
d h
formation of new combinations of
heritable material by the insertion of
nucleic acid molecules，produced by
whatever means outside the cell, into any
virus, bacterial plasmid or other vector
system so as to allow their incorporation
into a host organism in which they do not
naturally occur but in which they are
capable of continued propagation Also
propagation.
named gene cloning.

4. DNA Manipulation
•Gene cloning is production of many identical copies of the
same gene.
•If the inserted gene is replicated and expressed, we can
recover the cloned gene or protein product.
g p p
•Cloned genes have many research purposes:
determining the base sequence between normal and
mutated genes, altering the phenotype, obtaining the
mutated genes altering the phenotype obtaining the
protein coded by a specific gene, etc.
•Humans can be treated with gene therapy: alteration of
the phenotype in a beneficial way

5. DNA Manipulation
p
• Recombinant DNA (rDNA) contains DNA from two or more
different sources
– Requires:
• A vector
– introduces rDNA into host cell
– Plasmids (small accessory rings of DNA from bacteria) are
common vectors
– Phage vectors (bacterial viruses) can also be used
• Two enzymes to introduce foreign DNA into vector DNA
– A restriction enzyme - cleaves DNA
– Bacterial enzyme that stops viral reproduction by cleaving
viral DNA
– Act as molecular scisssors (cut p
( plasmids and foreign
g
human DNA)
– Produce short single stranded “sticky ends” where
insertions of foreign DNA can be made
– A DNA ligase enzyme - seals DNA into an opening created
g y p g
by the restriction enzyme
5

7. Cloning a
Human Gene
Restriction enzyme EcoRI
–Bacterial enzyme that stops viral reproduction by
cleaving viral DNA
–Act as molecular scisssors (cut plasmids and
foreign human DNA)
–Produce short single stranded “ ti k ends”
P d h t i l t d d “sticky d ”
where insertions of foreign DNA can be made
7

8. What is a Gene? a
Genome?
• A gene is a unit of inheritance. For
example,
example children tend to look like their
parents. We inherit our features through
our genes, half of which come from one
parent,
parent and half from the other.
other

9. • A gene is also defined by a digital code of
just four DNA b
j tf bases (A T G & C) th t is
(A,T,G that i
nearly universal for all known life forms,
whether viral, bacterial, fungal, plant,
animal or human. The average size gene
of bacteria is about 1,000 bases long.

10. • Since genes
are encoded
by the DNA
bases that
comprise th
i the
linear strands
of a chromos
chromos-
ome, the
genes are
arranged i
d in
linear order
along chrom-
g
osomes, and
they can be
mapped.
mapped

11. Why Clone DNA?
Wh
• A particular gene can be isolated and its
nucleotide sequence d t
l tid determined
i d
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be
investigated
g
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
resistance etc

12. How is DNA cloned?
Cell-based DNA cloning Cell-free DNA cloning (PCR)

13. Clone in dividing cells

14. Clone
in
P
C
R

15. Restriction Endonucleases
--The Molecular Scissors
Host enzymes that prevent the invasion of foreign
DNAs such as viral DNA by cutting them up
DNA, up.
Restriction
These enzymes cut within the foreign DNAs, rather
y g ,
than chewing them away from the ends.
Endonucleases
These enzymes recognize a specific DNA sequence
(4-12bp) which is twofold symmetry and cut both DNA
strands
Some enzymes make staggered cutsGAATTC
CTTAAG
CCCGGG
Some make even cuts
GGGCCC

17. Sticky end
Sticky end

19. DNA ligase covalently links two DNA strands
5’ 3’
Restriction
enzyme
Ligase
g
3
3’ 5
5’
Restriction
enzyme
Ligase

24. Plasmid: a cloning vector or
vehicle
h l

26. • Restriction Enzyme Mechanisms:
• Preparation of DNAs to be joined
• (a)Staggered cut: leaves “sticky
sticky
ends”

27. • Restriction Enzyme Mechanisms:
Preparation of DNA to be joined:
P ti f DNAs t b j i d
• (b) Blunt End

28. Ligation of DNA cut with a
Restriction E
R t i ti Enzyme
• Staggered “sticky ends”
sticky ends

29. Ligation of DNA cut with a
Restriction E
R t i ti Enzyme
• Role of T4 DNA Ligase

30. Selectable Markers:

32. cDNA Library
y
• Used to obtain
functional
eukaryotic
coding regions.
• E coli d
E. li does not t
process introns.
• First step:
Isolate poly A+
mRNA with oligo
(dT) cellulose

33. cDNA Library preparation

36. Cloning vectors
• Cloning vectors are carrier DNA molecules. Four important
features of all cloning vectors are that they:
f f ll l h h
• (i) can independently replicate themselves and the foreign
DNA segments they carry;
• (ii) contain a number of unique restriction endonuclease
cleavage sites that are present only once in the vector;
• (iii) carry a selectable marker (usually in the form of antibiotic
resistance genes or genes for enzymes missing in the host cell)
to distinguish host cells that carry vectors from host cells that
do not contain a vector; and
• (i ) are relatively easy to recover from the host cell.
(iv) l i l f h h ll

38. Plasmids
• Naturally occurring
extrachromosomal DNA
t h l
• Plasmids are circular dsDNA
• Plasmids can be cleaved by
restriction enzymes, leaving sticky
y , g y
ends
• Artificial plasmids can be
constructed by linking new DNA
fragments to the sticky ends of
plasmid

39. Vectors -- the DNA carriers
Must have a origin of replication
Allow the vector as well as the foreign DNA to amplify in the host cell
1) Plasmids
)
Origin of replication
2)
Antibiotic-resistant genes
Phage
g
Allow the host to grow on
s selective media
Can selectively amplify
this specific vector in the
host cell
Multiple cloning sites
Allow insertion of foreign DN

40. Plasmids

41. • Recombinant DNA vectors:
– Amplification of DNA fragment can be achieved in the
cell using cloning vectors: plasmid or bacteriophages
– Plasmid Small circular DNA in bacteria or yeast cells
Accumulate 1-5 kb inserts
LacZ encodes β galactosidase
β-galactosidase
Lacl – encodes factor controling
transcription of lacZ

42. Bacteriophage λ (Lambda)
B t i h (L bd )
• For cloning inserts of 10-20 Kb
g
• Plasmid libraries hold up to 10 kb
inserts

43. Bacteriophage λ (Lambda)Life
Cycle
C l
• Lytic
Cycle:Productio
n of progeny
• Lysogenic
Cycle:
l
Integration into
bacterial
b l
chromosome

44. BACs: Bacterial Artificial
Chromosomes
Ch
• Based on P1 bacteriophage, the F
p g ,
plasmid and the lacZ region of pUC
p
plasmids
• It’s a low copy number plasmid
• Carries 50 300kb fragments
50-300kb

45. BACs: Bacterial Artificial
Chromosomes

46. YACs:Yeast Artificial
Chromosomes
Ch

47. DNA can be inserted into a cell
by:
• Transformation
• El t
Electroporation
ti
• Protoplast
fusion

48. DNA can be inserted into a cell
by:
• Microinjection
• Gene gun

50. Bacterial transformation
Introduction of DNA into bacteria
Spontaneous uptake – low probability
E. coli – cells treated with CaCl2
Less than 1 of 103 cells acquire a
plasmid
Selection of transformed cells:
resistance to antibiotics
using chromogenic substances
Antibiotics: molecules produced by
microorganism that kill other
microorganism
peniciline, tetracycline, ciplroflaxine –
inhibits gyrase in the complex with DNA
– i hibit DNA replication
inhibits li ti
Chromogenic substances:

53. Screening
• The medium in this petri
p
dish contains the
antibiotic Kanamycin
• The bacteria on the right
contain Kanr, a plasmid
that is resistant to
Kanamycin,
Kanamycin while the one
on the left has no
resistance
• Note the difference in
growth

54. Propagation
p g
• Once colonies are
identified,
identified they are
cultured in broth to
increase numbers
and therefore the
amount of DNA
• Samples are also
prepared for storage
at -80 d
t 80 degrees. Th
They
can be kept for many
years this way
way.

55. Agricultural Applications
g pp
Herbicide
resistance
-Broadleaf plants
have been
engineered to be
resistant to the
herbicide
glyphosate
55

56. Agricultural Applications
Pest resistance
-Insecticidal proteins have been
transferred into crop plants to make
them pest-resistant
pest resistant
-Bt toxin from Bacillus thuringiensis
Golden rice
-Rice that has been genetically
Rice
modified to produce β-carotene
(provitamin A)
-Converted in the body to vitamin A
56

57. Agricultural Applications
g pp
Adoption of genetically modified (GM)
crops has been resisted in some areas
because of questions about:
-Crop safety for human consumption
C f t f h ti
-Movement of genes into wild
relatives
-Loss of biodiversity
y
57

58. Agricultural Applications
Biopharming
-Transgenic p
g plants are used to
produce pharmaceuticals
-Human serum albumin
Human
-Recombinant subunit vaccines
-Against N
A i t Norwalk and rabies
lk d bi
viruses
-Recombinant monoclonal
antibodies
-Against tooth decay-causing
bacteria
58

59. Transgenic Mammals
59

60. Medical Applications
• The insertion of genetic material
into human cells for the
treatment of a disorder
60

61. Recombinant DNA
Vaccines?
Strategy for a subunit vaccine for herpes simplex

62. Gene Therapy
Treatment of SCID (severe combined immunodeficiency). SCID affects the
( y)
maturation of immune cells that develop in bone marrow. SCID sufferers lack the
enzyme ADA (adenosine deaminase).
62