1. Recombinant DNA Technology
Presented by
Mona AL Boreikan

2. What is recombinant DNA?
● Recombinant DNA is the formation of a novel
DNA sequence by the formation of two DNA
strands.
● These are taken from two different organisms.
● These recombinant DNA molecules can be
made with recombinant DNA technology.

3. What is recombinant DNA technology?
The procedure is to cut the DNA of the donor
organism into pieces with restriction
enzymes, and insert one of these fragments into
the DNA of the host.

4. Recombinant DNA technology
=
Genetic engineering
=
Gene cloning

5. The History of Recombinant DNA Technology
• 1970 Hamilton Smith, at Johns
Hopkins Medical School, isolates
the first restriction enzyme, an
enzyme that cuts DNA at a very
specific nucleotide sequence.
Over the next few years, several
more restriction enzymes will be
isolated.
• 1972 Stanley Cohen and Herbert
Boyer combine their efforts to
create recombinant DNA. This
technology will be the beginning
of the biotechnology industry.

6. The History of Recombinant DNA Technology
• 1976 Herbert Boyer
cofounds Genentech, the
first firm founded in the
United States to apply
recombinant DNA
technology.
• 1978 Somatostatin, which
regulates human growth
hormones, is the first
human protein made
using recombinant
technology.

7. What Required for Recombinant DNA Technology
First ; Cloning Vectors
A cloning vector is
a small piece of
DNA into which a
foreign DNA fragment
can be inserted.

8. What are the kinds of Cloning Vectors
1- Plasmid Cloning Vectors
2- Bacteriophage Vectors
3- Cosmids Vectors
4- (BACs)
5- YACs

9. 1- Plasmid Cloning Vectors
• A plasmid is a DNA molecule
that is separate from the
chromosomal DNA.
• It can replicate
independently of the
chromosomal DNA.
• It is double-stranded and, in
many cases, circular.
• Plasmid sizes vary from 1 to
over 1,000 kilobase pairs
(kbp), but it can only contain Illustration of a bacterium with plasmid
inserts of about 1–10 kbp. enclosed showing chromosomal DNA and
plasmids

10. 1- Plasmid Cloning Vectors
• Origin of replication (ORI).
• Plasmid is used to multiply (make many copies of) or express
particular genes.
• Plasmid containing genes that make cells resistant to particular
antibiotics, Selectable marker(s)
• Plasmid containing a multiple cloning site (MCS, or polylinker).

11. 1- Plasmid Cloning Vectors
How are the plasmids inserted into bacteria?
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as
with the top instance; whereas episomes, the lower example, integrate into the host chromosome

12. 1- Plasmid Cloning Vectors
Cloning DNA into a Plasmid to Produce Recombinant DNA

13. 2- Bacteriophage Vectors
● Bacteriophage is any one
of a number of viruses
that infect bacteria.
● Commonly based upon l
phage. Lambda phage is a
virus particle consisting of
a head, containing
double-stranded linear
DNA as its genetic
material, and a tail that
can have tail fibers.
The structure of a typical
tailed bacteriophage

14. 2- Bacteriophage Vectors
● Most internal genes deleted.
● Insert DNA into middle region (up to10-15 kb)
● The phage genes expressed in lysogenic cycle code for proteins
that repress expression of other phage genes.

15. 2- Bacteriophage Vectors
How the DNA phage inserted into bacteria?
The lysogenic and Lytic cycle.

16. 2- Bacteriophage Vectors
Cloning DNA into Bacteriophage to Produce Recombinant DNA

17. 3- Cosmids Vectors
• Cosmid type of hybrid plasmid.
• Plasmid with λ phage packaging sequence (cos)
• Cosmids are able to contain 37 to 52 kb of DNA.
• Packaged into λ particles and injected into host cells.
• Circularizes in cell and continues as a large plasmid

18. 3- Cosmids Vectors
Cloning DNA by cosmid to Produce Recombinant DNA

19. 3- (BACs) Vectors
• Bacterial artificial
chromosome.
• Can clone up to 200 kb
DNA fragments.
• Based upon F plasmid.
• A similar cloning
vector, called a PAC has
also been produced from
the bacterial P1-plasmid.
• Origin, selectable
marker, promoters to
expressed cloned genes

20. 3- (BACs) Vectors
How the
BAC
inserted
into bacteria
to Produce
Recombinant
DNA?

21. 4 - (YACs) Vectors
• Yeast artificial
chromosomes
• Have
centromere, telome
res and an origin of
replication, plus
selectable markers
• Cloned segments of
250 kb

22. 4 - (YACs) Vectors
How the
YAC
inserted
into yeast to
Produce
Recombinant
DNA?

23. Summary of vectors and what they can carry.
The size of DNA that vector
Vector
can carry
0 – 10 kb Standard plasmid
0 – 23 Kb Lambda Bacteriophage
30 – 44 Kb Cosmid
70 – 100 Kb Bacteriophage P1
130 – 150 Kb P1 Artificial chromosome PAC
Maximum 300 Kb Bacterial Artificial Chromosome BAC
0.2 – 2 Mb Yeast Artificial Chromosome YAC
kb (= kbp) = kilo base pairs = 1,000 bp
Mb = mega base pairs = 1,000,000 bp

24. 5 - Expression Vectors
• Also known as an
expression
construct
• It include
regulatable high
level expression
promoter
– T7 phage
promoter
– lac operator
– lac repressor
gene

25. The three most important signals for Expression.

26. What Required for Recombinant DNA Technology
Second; Enzymes
A- Restriction enzyme
• OR restriction endonuclease) is an enzyme that cuts double-
stranded or single stranded DNA at specific recognition
nucleotide sequences known as restriction sites.

27. Summary of some kinds of Restriction
enzymes and their cut sites.

28. Second; Enzymes
B - A DNA ligase enzyme
• Catalyse the joining or recombining of DNA fragments
(ligation).
DNA ligase Ligation by DNA ligase enzyme

29. What the steps for recombinant DNA technology?
• Protocol;
– Isolate target DNA
– Cut with RE
– Ligate to vector
– Transform to host
cells
– Plate on antibiotic-
containing medium
– Identify recombinant
plasmids
– Identify/characterize
specific clones

30. What the steps for recombinant DNA technology?

31. Examining the results of a
restriction digest ( after recombinant )
After treatment with a
restriction
endonuclease,
the resulting DNA
fragments can be
examined
by agarose gel
electrophoresis to
determine
their sizes and to make An agarose gel plate. The hybridized DNA
shore if recombinant strands show a pink light because of the
DNA happened or not. binding of the fluorescent labeled probe.

32. What is Gel Electrophoresis
• A technique used to separate DNA fragments by
size
• The gel (agarose or polyacrylamide) is subjected to
an electrical field
• The DNA, which is negatively-charged, migrates
towards the positive pole.
• The larger the DNA fragment, the slower it will
move through the gel matrix.
• DNA is visualized using fluorescent dyes.

34. Other types of

35. Reproductive Cloning
* Reproductive cloning is
performed with the
express intent of creating
another organism.
* This organism is the exact
duplicate of one that
already exists or has existed
in the past.
* Cloning of
plants, animals, and
humans falls into the class
of reproductive cloning

36. How is Reproductive Cloning Performed?
* It is performed using a technique called Somatic Cell Nuclear Transfer (SCNT).
* The genetic material from a donor egg is removed, so that you are left with an
empty egg.
* Then, a cell is taken from the organism to be cloned and its nucleus is removed.
* This nucleus is then transferred into the empty donor egg.

37. What Is Reproductive Cloning Used For?
Reproductive cloning has only
been used for research
purposes.
Reproductive cloning could be
used effectively for
repopulating endangered
species or to help make
breeding of specific animals
easier.
Reproductive cloning uses could
also include the production of
organisms with specific
characteristics, such as drug-
producing animals or
genetically "unique" animals.

38. Dolly was created in a process called
“Somatic Cell Nuclear Transfer”
Professor Ian Wilmut is now a Sir for his work creating the world’s first cloned
mammal in 1996 - 2003

39. Therapeutic Cloning
• It is performed, not to
produce another
organism, but to harvest
embryonic stem cells for
use in medical treatments.
• Embryonic stem cells are
those cells found inside of
developing embryos.
• They can be used to
produce a number of
different cells including
tissue, muscle, and organ
cells.

40. How is Therapeutic Cloning Performed?
* A cell is removed from the patient requiring medical treatment.
* The nucleus of this cell is removed and inserted into an empty donor egg.
* Division is encouraged through the use of special chemicals or an electric
current.
* The resulting embryonic stem cells are then removed from this embryo and
used to treat the patient.

41. What is Therapeutic Cloning Used For?
• Therapeutic cloning is intended for medical use.
• The embryonic stem cells that this type of cloning produces can be
used to create skin for burn victims, organs for transplant patients, or
cells for those with spinal cord injuries.

42. What is Therapeutic Cloning Used For?
• And because the cells come from the patient herself, there are
no issues of cell rejection.
• Therapeutic cloning may also help those suffering from heart
disease, Alzheimer's Disease, or Parkinson's Disease.

43. Can organs be cloned for use in transplants?
 Scientists think that therapeutic
cloning can be used to make
tissues and organs for transplants.
 To do this, DNA would be taken
from the person who needs the
transplant and put in a egg. After
the egg with the patient's DNA
starts to divide, embryonic stem
cells that can be transformed into
any type of tissue would be
harvested..
 Stem cells would generate an
organ or tissue that is a genetic
match to the recipient, as a result
organ donation would reduce
significantly.

44. Risks of Cloning
 More than 90% of cloning attempts fail to produce viable
offspring
 Cloned animals tend to have higher rates of infection tumor
growth, and other disorders.
 Clones have been known to die mysteriously.

45.  Because of the lack of
understanding about
reproductive cloning
scientists believe it is
unethical to attempt
to clone humans.
 About 30% of clones
born alive are
affected with “large
offspring syndrome”

46. • Several cloned
animals have died
prematurely from
infections or other
complications, the
same problems would
be expected in human
cloning
• The attempt to clone
humans at this time is
considered
potentially dangerous,
and ethically
irresponsible.

47. The End