Genetic Engineering is a set of techniques capable to allow the identification, manipulation and multiplication of genes of living organisms.

1. Genetic Engineering and
Recombinant DNA Technology
Presented by
Dr. B.Victor., Ph.D.
Email: bonfiliusvictor @gmail.com
Blog: bonvictor.blogspot.com

2.  Genetic engineering-
definition, objectives and basic
Presentation methodology
out line  Recombinant DNA technology-
definition, objectives, history and
enzyme tools.
 Restriction enzymes - features, types
of cuts.
 Restriction mapping- applications
 Vector DNA – types, characteristics
and practical features
 Host cell types
 Recombinant DNA technology-
procedure
 Gene transfer technology
 Screening technology
 Applications

3. Genetic engineering
Definition -1
• A set of techniques capable to allow the
identification, manipulation and multiplication of
genes of living organisms.
Definition -2
• Changing of genes by using in vitro processes.
Other names
• Gene manipulation, gene cloning
• Recombinant DNA technology, genetic modification

4. Objectives of Genetic Engineering
Basic research on gene
structure and function
Production of useful products
and services
Generation of transgenic
plants and animals
Investigation of human
genome for gene therapy

5. Genetic
engineering
Basic methodology

6. Recombinant DNA technology
r DNA technology
• The technology of preparing
r DNA in vitro by cutting up
DNA molecules and splicing
them together fragments from
more than one organism.

7. Meaning r DNA technology
Modifying the genetic
make up an organism
by:
• Adding new genes
• Changing the existing
genes

8. Objectives of  Artificially synthesize new
r DNA genes.
Technology
 Altering the genome of an
organism.
 Bring about new gene
combinations not found in
nature.
 Understanding the
hereditary diseases and their
cure.
 Improving human genome.

9. Discovery of DNA structure-
Watson and Crick in 1953
Isolation of DNA ligase in 1967
History of
recombinant
DNA Isolation of REase in 1970
technology
Jackson, Symons, and Berg (1972)
generated first recombinant DNA
molecules.
Cohen and Boyer (1973)
produced first plasmid vector
capable of being replicated within
a bacterial host

10. Biological tools of r DNA technology
Foreign DNA/ Vehicle/vector Culture media,
Enzymes Host cells
Synthetic DNA DNA Buffers, reagents

11.  DNA or RNA polymerase-
Chemical replicating or annealing a DNA
knives in chain.
molecular  Reverse transcriptase – synthesize
carpentry c DNA from RNA template.
Enzymes are  DNA ligase – joining DNA strands
chemical knives together.
in r DNA  Nuclease-breaks phospho-diester
technology bonds within free ends
(exonucleases) or in an interior
position( Endonucleases ).
 Restriction endonuclease –
recognize a specific base sequence
and cuts the DNA.

12.  A special class of sequence –
Restriction
specific enzymes
endonucleases
 Found in bacteria which protect its
(RE ases)
genetic material from the invasive
attacks of viruses.
 Site-specific - cleave DNA molecules
only at specific nucleotide
sequences.
 REases recognize DNA base
sequences that are palindromes.
 REases make two single stranded
breaks, one in each strand.
 RE ases make staggered cuts with
complementary base sequences for
easy circularization.

13.  Recognition sequences of RE ases
are palindromes
Restriction
 A palindrome is a word, phrase,
Enzymes- number or other sequence of units
Types of cuts that can be read the same way in
either direction
 Cohesive (sticky) ends –
overhanging single-stranded ends
 Blunt ends – double-stranded,
non-overhanging ends

14. Salient features
Bacterial
of Restriction Endonucleases
enzymes
endonucleases
(RE ases)
Different REases Names of REases
Isolated from are derived
different from source
bacteria bacteria.
Cut DNA into Basic tool of
defined and Genetic
reproducible
fragments Engineer

15.  A map showing the unique sites
Restriction of cutting of the DNA of a
mapping particular organism by a single
REase enzyme.
 A particular REase generates a
unique set of DNA fragments
with specific base sequence.
 Another enzyme will generate a
different set of DNA fragments
from the same DNA molecule.
 The family of DNA fragments
generated by a single enzyme
can be detected easily gel
electrophoresis.

16.  Preparing a restriction map
Applications of
of DNA
Restriction
endonucleases  Fragmenting genomic DNA
(RE ases) prior to Southern Blotting.
 Generating DNA fragments
RE ases catalyze that can be sub-cloned in
sequence –
appropriate vectors.
dependent double-
stranded breaks in  Generating DNA fragments
DNA yielding a for labeled probes
homogeneous
population of DNA
fragments.

17. Vector DNA or Plasmid DNA
Vehicle DNA
Bacterial Bacteriophage
DNAs DNA
The DNA which
Vector
acts as a carrier is DNA
a vehicle DNA
Viral DNAs Yeast DNAs

18.  Types of Vectors
Types of  Bacterial plasmid vectors
vectors  Bacteriophage vectors
 Cosmid vectors
 Expression vectors
 Bacterial Artificial Chromosomes
(BAC)
 Yeast Artificial Chromosomes
(YAC)
 Ti and Ri vectors

19.  Capable of autonomous
replication independent of the
Characteristics main bacterial chromosome
of an
prokaryotic  Easy to isolate, i.e. small.
vector  Non -toxic to host cells.
 Have space for foreign inserts.
 Have unique restriction sites
for common restriction
enzymes.
 Have convenient markers for
selection of transformants, e.g.
antibiotic resistance genes
 Be relaxed, i.e. multiple copies
in a host cell.

20.  Size
 Origin of replication (ori)
 Multiple cloning site (MCS)
Practical  Selectable marker genes
Features of
 RNA polymerase promoter
DNA Cloning
sequences
Vectors
 DNA sequencing primers

21.  Plasmid DNA – small
circular DNA found in
Plasmids bacteria
 They replicate
autonomously.
 Easily purified
 Confer antibiotic resistance
to host bacteria –allow easy
identification.
 First type of cloning vector
developed.

22.  Two types of phage vectors
Phage Vectors have been extensively
developed-λ and M13.
 phage vectors have engineered
phage genomes previously
genetically modified to include
restriction sites.
 after insertion of foreign
DNA, the recombinant phage
genome is packaged into the
capsid and used to infect host
cells

23. 23
 Hybrid vector constructed to
contain features from both
Cosmids phages and plasmids.
 Cosmids have a selectable
marker, multiple cloning
sites from plasmids and a
cos site from l phage

24. 24
 large fragments of DNA can
Artificial be cloned.
Chromosomes
 Mapping of genes is easier.
 One copy of YAC is present
per cell.
 yeast artificial chromosomes
(YACs)
 bacterial artificial
chromosomes (BACs)
 played important role in the
human genome project

25. Host cell types
Host cell types
Prokaryotic Eukaryotic
hosts hosts
Yeast
Bacteria
Algae
E . Coli fungi
Bacillus sp.
Yeast - Saccharomyces
Pseudomonas sp.
Fungi- Aspergillus, Neurospora
Streptomyces sp. Algae - Chlamydomonas

26. Two types of host-vectors
Cloning
• Propagation of
vector DNA inserts
Expression
• Production of
vector proteins

27. Isolate desired
DNA
Making of
Cut with a suitable
r DNA REase
Ligate into a suitable
cloning vector
Transform r DNA
into a suitable host
cell

28. Step 1– fragmentation -
breaking apart a strand of
DNA
Molecular
Cloning /
DNA cloning Step 2 – ligation-gluing
together pieces of DNA in a
desired sequence.
Molecular cloning
refers to the Step 3 –Transfection -
process of making inserting the newly formed
DNA into cells.
multiple DNA
molecules.
Step 4-Screening / selection –
selecting out the cells that
were successfully tranfected
with the new DNA

29. Recombinant DNA cloning procedure
Screening of Fine
Foreign
Transfection transformants chemicals
DNA
Hormones
Host Desired
r DNA Enzymes
cell products
vaccines
Plasmid
Antibiotics
DNA
Antibodies
Blood factors

30. 1. Choice of host organisms
and cloning vector
DNA
2. Preparation of vector DNA
cloning
3. Preparation of DNA to be
protocol – cloned
7 steps
4. Creation rDNA.
5. Introduction of rDNA into
the host organism.
6. Selection of organisms
containing rDNA.
7. Screening for clones with
desired DNA inserts and
biological properties.

31. Gene transfer technology
Transduction • Virus mediated gene transfer
Tranfection • Chemical or physical tricks to persuade cells to
take DNA from the culture medium
• Physically inserting the gene
Direct transfer • e.g. microinjection
Natural gene • A receptor – mediated lateral binding
• Fusogenic proteins used
transfer

32.  This method was described by
Graham and Van der Eb in 1973.
Calcium  A process for inserting foreign
phosphate – DNA into bacteria
co precipitate  Treat bacterial cells with ice-cold
method calcium chloride
 Add plasmid DNA to cells chilled
on ice which form calcium
Transformation of phosphate –DNA precipitate.
Bacterial Cells  Heat the cell and DNA mixture to
42oC
 Membrane becomes fluid and
plasmid DNA enters bacterial cells
and is replicated and expressed

33.  It involves a brief application of
high voltage electric current to
Electroporation the cells resulting in the
formation of transient holes in
the cell membrane through which
plasmid DNA can enter the cell.
 The transformation efficiency is
high.
 Quick restoration of membrane
fluidity and closing of pores is
crucial for survival of cell after
the pulse.

34. Selection
techniques for DNA
rDNA Colony
hybridization
molecules immunoassay
assay
Screening by Genetic
protein screening
activity methods

35.  After transformation, the
Process of bacteria are challenged with
selection an antibiotic (such as
ampicillin).
 If the E. coli have taken up
and expressed an ampicillin
resistance gene on a
Selection is a plasmid, they will live -
process designed to otherwise they will die.
facilitate the
identification of  This process is called
recombinant bacteria selection because selected
while preventing the
growth of non- bacteria may survive.
transformed
bacteria.

36.  This technique was
introduced by Grunstein and
DNA
hybridization
Hogness (1978).
assay  The target DNA is denatured
at 800C and bound to a
nitrocellulose filter discs.
 Such filters are hybridized
with radioactive DNA
probes.
 The results are monitored by
autoradiography.

37.  The transformed colonies are
transferred to a nitrocellulose filter.
Colony  The colonies are lysed and the released
immunoassay proteins are attached to the matrix.
 The matrix is treated with a primary
antibody which specifically binds to the
proteins encoded by the target gene.
 Then the matrix was washed to remove
any unbound antibody. Then the
matrix was treated with a second
antibody which was an
enzyme, alkaline phosphatase.
 the target protein (antigen)was treated
with a colorless substrate.
 The colorless substrate is hydrolysed
by the alkaline phosphatase into a
colored complex.

38. The Tools of  Gene Libraries
Recombinant  Collections of cloned DNA
DNA Technology fragments from a particular
organism contained within
bacteria or viruses as the host
 Library may contain all genes of a
single chromosome
 Screening, identification and
characterization of cloned
fragments are possible with
suitable probes.
© 2012 Pearson Education Inc.

39.  contains only complementary DNA
molecules synthesized from mRNA
molecules in a cell.
cDNA  mRNA from tissue of interest is
Libraries isolated
 Converted to a double-stranded
DNA by using the enzyme reverse
transcriptase
 Called complementary DNA
(cDNA) because it is an exact copy
of the mRNA

40.  Multiplying DNA in vitro: The
Techniques of Polymerase Chain Reaction
Recombinant (PCR)
DNA  Developed in the 1980s by Kary Mullis
Technology  Technique for making copies, or
amplifying, a specific sequence of DNA
in a short period of time
 Repetitive process consisting of three
steps
 Denaturation
 Priming
 Extension
 Can be automated using a
thermocycler
 At the end of one cycle, the amount of
DNA has doubled
 Cycles are repeated 20–30 times
© 2012 Pearson Education Inc.

41.  Separating DNA Molecules: Gel
Techniques of
Recombinant
Electrophoresis
DNA Technology  Gel electrophoresis
 Separates molecules based on
electrical charge, size, and shape
 Allows to isolate DNA of interest
 Negatively charged DNA drawn
toward positive electrode
 Agarose makes up gel; acts as
molecular sieve
 Smaller fragments migrate faster
than larger ones
 size is determined by comparing
distance migrated to standards
© 2012 Pearson Education Inc.

42. Techniques of  Separating DNA Molecules:
Recombinant Gel Electrophoresis and the
DNA Technology Southern Blot
 Southern blot
 DNA transferred from gel to
nitrocellulose membrane
 Probes used to localize DNA
sequence of interest
 Uses of Southern blots
 Genetic “fingerprinting”
 Diagnosis of infectious disease
© 2012 Pearson Education Inc.

43. Techniques of  DNA Microarrays
Recombinant  Consist of molecules of
DNA Technology immobilized single-stranded DNA
 Fluorescently labeled DNA washed
over array will adhere only at
locations where there are
complementary DNA sequences
 Uses of DNA microarrays
 Monitoring of gene expression
 Diagnosis of infection
 Identification of organisms in an
environmental sample
© 2012 Pearson Education Inc.

44.  In situ hybridization
 Used to determine the cell type that
Applications is expressing the mRNA
of  Tissue of interest is preserved in a
Recombinant fixative solution and embedded in
a wax-like substance
DNA
 Tissue can be sliced into very thin
Technology - sections attached to microscope
Studying Gene slides
Expression  Slides are incubated with a probe
to the gene of interest
 Probe hybridizes with mRNA in
cells
 Probe is detected

45.  Studying Gene Expression
 Gene microarrays
 DNA microarray analysis
 Single-stranded DNA molecules
Applications of
are attached onto a slide using a
Recombinant robotic arrayer fitted with tiny pins
DNA  Can have over 10,000 spots of DNA
Technology  Extract mRNA from tissue of
interest, tag it with fluorescent
dye, and incubate overnight with
the slide
 mRNA will hybridize to spots on
the microarray that have
complimentary DNA sequences
 Slide is scanned with a laser that
causes the spots to fluoresce

46. Applications of  Genetic Mapping
Recombinant  Provides knowledge of total
DNA Technology
number of all genes.
 Shows relationships between
genes.
 Provides all genetic
information about the
organism.
© 2012 Pearson Education Inc.

47. Applications of  Pharmaceutical and
Recombinant Therapeutic Applications
DNA Technology  Gene therapy
 Missing or defective genes replaced
with normal copies
 Medical diagnosis
 Patient specimens can be examined
for presence of gene sequences
unique to certain pathogens
 Xenotransplants
 Animal cells, tissues, or organs
introduced into human body
© 2012 Pearson Education Inc.

48.  Agricultural Applications
Applications of
Recombinant  Production of transgenic organisms
DNA Technology  Recombinant plants and animals
altered by addition of genes from
other organisms
 Herbicide tolerance
 Gene from Salmonella conveys
resistance to glyphosate (Roundup™)
Farmers can kill weeds without
killing crops
 Salt tolerance
 Scientists have inserted gene for salt
tolerance into tomato and canola
plants
 Transgenic plants survive, produce
fruit, and remove salt from soil
© 2012 Pearson Education Inc.

49. Applications of
 Agricultural Applications
Recombinant  Pest resistance
DNA Technology  Bt toxin
 Naturallyoccurring toxin
harmful only to insects
 Gene for Bt toxin inserted into
various crop plants
 Genes for Phytophthora resistance
inserted into potato crops
© 2012 Pearson Education Inc.

50. Applications of
 Agricultural Applications
Recombinant  Improvements in nutritional value
DNA Technology and yield
 Tomatoes allowed to ripen on vine
and shelf life increased
 Genefor enzyme that breaks
down pectin suppressed
 BGH allows cattle to gain weight
more rapidly
 Produce meat with lower fat
content and produce 10% more
milk
 Gene for β-carotene inserted into
rice
© 2012 Pearson Education Inc.

51.  Started in 1990 by the U.S.
Department of Energy
The Human  International collaborative effort
Genome to identify all human genes and to
sequence all the base pairs of the
Project 24 human chromosomes
 20 centers in 6 countries:
China, France, Germany, Great
Britain, Japan, and the United
States.
 April 14, 2003, map of the human
genome was completed
 Consists of 20,000 to 25,000
protein-coding genes

52. Medical products made from rDNA tech
Product year treatment
Human insulin 1982 diabetes
somatotropin 1985 Pituitary dwarfism
Hepatitis B vaccine 1986 Immunization for hepatitis B virus
Erythropoietin 1988 anemia
Interleukin-2 1989 Cancer of kidney
Whooping cough vaccine 1989 Immunization for Whooping cough
Factor VIII 1993 hemophilia

53.  Dr.B.Victor is a highly
experienced professor, recently
retired from the reputed
educational institution- St.
Xavier’ s
About the College, Palayamkottai, India-
Presenter 627001.
 He was the dean of
sciences, IQAC coordinator and
assistant controller of
examinations.
 He has more than 32 years of
teaching and research
experience
 He has taught a diversity of
college courses and guided 12
PhDs.
 Send your comments to :
bonfiliusvictor@gmail.com

54. Thank a lot for your watching