An introduction about recombinant DNA as a tool for genetic engineering.

1. Genetic Engineering
Recombinant DNA
Hala Abuzied
MS Human Genetics
Pharmacist/ Research Fellow
Alexandria University Cancer Research Cluster

2. Genetic Engineering
• Also called Genetic modification or Genetic manipulation
• It is the process of altering of an organism's genes using
biotechnology. It is a set of technologies used to change the
genetic makeup of cells.
• This may mean:
- Changing one base pair (A-T or C-G)
- Deleting a whole region of DNA
- Introducing an additional copy of a gene.
- Extracting DNA from another organism’s genome and combining it
with the DNA of another individual.
• Genetic Engineering can be applied to any organism, from a virus
to a sheep.
• Genetic Engineering is of value in many Research and medical
areas

3. Genetic Engineering
• The term genetic engineering is often thought to be rather
vague term, yet it is probably the label that most people
would recognize.
• However, there are several other terms that can be used to
describe the technology, including
- Gene cloning
- Genetic modification
- Gene editing
- Recombinant DNA technology

4. Genetic Engineering
• Genetic engineering involves the use of Recombinant DNA
technology.
• Recombinant DNA technology: the process by which a DNA
sequence is manipulated in vitro, thus creating recombinant
DNA molecules that have new mix of genetic material.
• The recombinant DNA is then introduced into a host organism.
• If the DNA that is introduced comes from a different species,
the host organism is now considered to be transgenic.

5. Genetic Engineering
Bacterial strain that produces human insulin:
• One example for: transgenic microorganism, Recombinant
DNA and Genetic engineering.
• The insulin gene from humans was inserted into  plasmid.
• This recombinant DNA plasmid was then inserted  bacteria.
• As a result, these transgenic microbes are able to produce and
secrete human insulin.

6. Genetic Engineering
• Plasmids are mobile pieces of DNA (often circular) that bacteria
can easily trade amongst themselves or simply acquire from the
environment.
• Many prokaryotes are able to acquire foreign DNA and
incorporate functional genes into their own genome through:
- Conjugation: “mating” with other bacterial cells
- Transduction: viral infection
- Transformation: uptake of DNA into bacterial, yeast or plant
cells
• These mechanisms are examples of horizontal gene transfer
the transfer of genetic material between cells of the same
generation.

8. Genetic Cloning
• Set of methods used to construct
recombinant DNA and incorporate
it into a host organism:
“ Tool for making recombinant DNA”
- Restriction Enzymes and Ligases
- Plasmids
- Molecular Cloning using
Transformation
- Molecular Cloning Using
Conjugation
- Molecular Cloning Using
Transduction

9. Restriction Enzymes & Ligases
• Restriction endonucleases: bacterial enzymes produced as a
protection mechanism to cut and destroy foreign cytoplasmic
DNA that is result of bacteriophage infection.
• Use for cutting DNA fragments that can then be introduced to
another DNA molecule to form recombinant DNA molecules
• Each restriction enzyme cuts DNA at a characteristic recognition
site : DNA sequence typically between 4 to 6 base pairs in length

10. Restriction Enzymes & Ligases
• A restriction enzyme recognizes the DNA recognition site and cuts
each backbone at identical position, forming “DNA sticky ends” in
both whole DNA and Plasmids
• Molecules with complementary sticky ends easily anneal and form
hydrogen bonds between complementary bases at their sticky ends.
• The annealing step allows forming new hybrid double strand DNA.
• ligation by DNA ligase can then rejoin the two sugar-phosphate
backbones of the DNA.
• Restriction enzymes are named after the bacteria from which they
were isolated.

11. Restriction Enzymes
• After cutting DNA with restriction enzymes, the fragments can be
separated on an Agarose Gel.
• The smaller fragments will migrate further than the longer fragments
in an electric field.
• The bands are compared to standard DNA of known sizes. This is
often called a DNA marker, or DNA ladder.
• After analyzing your results, you draw a restriction map of the cut sites.
• A restriction map is a diagram of DNA showing the cut sites of a
series of restriction enzymes.

12. Restriction Enzymes

13. Restriction Enzymes

14. Plasmids
• Small pieces of typically circular, double-
stranded DNA that replicate independently
of the bacterial chromosome.
• Genes of interest after restriction are
inserted into plasmids
• Used as vectors: DNA molecules that carry
DNA fragments from organism to another.
• Vectors can be genetically engineered to
have specialized properties ex plasmid
vectors contain genes that is antibiotic
resistance allow researchers to easily find
plasmid containing colonies by plating
them on media containing the
corresponding antibiotic.

15. Plasmids
• Plasmid vectors used for cloning typically have a polylinker site,
or multiple cloning site (MCS): a short sequence containing
multiple unique restriction enzyme recognition sites that are used
for inserting DNA into the plasmid after restriction digestion of
both the DNA and the plasmid.

16. Molecular Cloning using
Transformation
• Transformation: a process in which bacteria take up free DNA
from their surroundings. In nature, free DNA typically comes
from other lysed bacterial cells; in the laboratory, free DNA in the
form of recombinant plasmids.
• Bacillus spp: are naturally competent, they are able to take up
foreign DNA. Other bacteria are not naturally competent.
• In most cases, bacteria must be made artificially competent
increasing the permeability of the cell membrane:
- Using chemical treatments that neutralize charges on the cell
membrane
- Exposing the bacteria to an electric field that creates microscopic
pores in the cell membrane.

17. Molecular Cloning using
Transformation
• Following the transformation protocol  bacterial cells are plated
onto an antibiotic-containing medium to inhibit the growth of the
many host cells that were not transformed by the plasmid with the
antibiotic resistance property, so will be able to separate the
plasmids with the inserted DNA

18. Molecular Cloning Using
Conjugation
• F plasmids “Fertility plasmids” are transferred between
bacterial cells through the process of conjugation.
• F plasmids encode a surface structure called an F pilus that
facilitates contact between bacterial cells
• Cytoplasmic bridge between the two cells and the F-plasmid-
containing cell replicates its plasmid  transferring a copy of
the recombinant F plasmid to the recipient cell  can produce
its own F pilus

19. Molecular Cloning Using
Conjugation

20. Molecular Cloning Using
Transduction
• Bacteriophages can be used to
introduce recombinant DNA into host
bacterial cells through a manipulation
of the transduction process.
• In the laboratory, DNA fragments of
interest can be engineered
into Phagemids.
• Phagemids: plasmids that have phage
sequences that allow them to be
packaged into bacteriophages.
• Bacterial cells can then be infected with
these bacteriophages so that the
recombinant phagemids can be
introduced into the bacterial cells.

21. Molecular Cloning Using
Transduction

22. Genomic Libraries
• The library is a nearly complete copy of an
organism’s genome contained as
recombinant DNA plasmids engineered into
unique clones of bacteria.
• Having such a library allows a researcher to
create large quantities of each fragment by
growing the bacterial host for that fragment.
• Genomic Libraries can be used to
determine:
- Sequence of the DNA
- Function of any genes present

23. Genomic Libraries (DNA)
• One method  is to ligate individual restriction enzyme-
digested genomic fragments (Whole genome) into plasmid
vectors cut with the same restriction enzyme.
• After transformation into a bacterial host, each transformed
bacterial cell takes up a single recombinant plasmid and
grows into a colony of cells.

24. Genomic Libraries (cDNA)
• Genomic libraries for expressed genes
in an organism  use the organism’s
messenger RNA (mRNA).
• All organism have the same genomic
DNA in all cells  different tissues
express different genes  producing
different mRNA.
For example: all human cells’ genomic
DNA contains the gene for insulin, but
only cells in the pancreas express mRNA
directing the production of insulin.
Because mRNA cannot be cloned
directly.

25. Genomic Libraries (cDNA)
• mRNA  using reverse transcriptase make cDNA (cDNA) 
used by DNA polymerase to make  double strand DNA
copies these fragments ligated into plasmid vectors or
bacteriophage to produce a cDNA library.

26. Sequencing
• Sequencing – determining the order and arrangement of
G’s, A’s, T’s and C’s in a segment of DNA.

27. Applications of RecombinantDNATechnology

28. References:
• Desmond S. T. Nicholl, “An Introduction to Genetic
Engineering”volume 2, 3 ed, 2008.
• Raylene Ramos Moura, Luciana Magalhães Melo, and Vicente
José de Figueirêdo Freitas. "Production of Recombinant
Proteins in Milk of Transgenic and Non-Transgenic
Goats." Brazilian Archives of Biology and Technology 54 no. 5
(2011): 927–938.
• William S.M. Wold and Karoly Toth. "Adenovirus Vectors for
Gene Therapy, Vaccination and Cancer Gene Therapy." Current
Gene Therapy 13 no. 6 (2013): 421.

29. Thank You