This presentation is as per the curriculum of B.Sc-III Sem-VI Unit-V subject Botany of SGBAU Amravati

1. GENETIC ENGINEERING
B. Sc-III
Semester-VI
Mr. Kailash S. Sontakke
Assistant Professor
Dept. of Botany, G.S.G.College, Umarkhed.
sontakke@gsgcollege.edu.in

2. Genetic Engineering.
• Allaby (1995) has defined The genetic engineering as the
modification of the genetic information of living organisms by
direct manipulation of their DNA
• rDNA Technology is the technique of manipulating the genome of a
cell or organism so as to change the phenotype desirably.
• also called gene cloning, recombinant DNA technology or gene
manipulation

3. The Basic principal of
recombinant DNA technology

4. Tools used in recombinant DNA technology
• 1. Enzymes such as exonucleases, endonucleases, restriction enzymes (=restriction
endonucleases), SI enzymes (to change cohesive ends of single stranded DNA
fragments into blunt ends), DNA ligases, alkaline phosphatase, reverse transcriptase,
DNA polymerases.
• 2. Foreign DNA/Passenger DNA/Gene of Interest. It is a fragment of DNA
molecule which is enzymatically isolated and cloned. The gene is identified on a
genome and pulled out from it either before or after cloning. The cloned foreign
DNA fragment expresses normally as in parent cell.
• 3. Host

5. Tools used in recombinant DNA technology
• 4. Cloning vectors. Vectors or vehicle DNA are those DNA that can carry a
foreign DNA fragment when inserted into it. Based on the nature and sources, the
vectors are grouped into bacterial plasmids, bacteriophages, cosmids and phasmids.
• 5. cDNA bank or cDNA library.
• 6. Genomic library. Gene bank or genomic library
• 7. Molecular probes. A probe is either a radioactive labelled (32 P) or
nonradioactive labelled (viz., biotin or digoxigenin), single stranded nucleic acid (20–
40 nucleotide long) with a sequence complementary to at least one part of the
desired DNA.

6. Techniques used in recombinant DNA technology
1.DNA/ RNA Extraction
2. Electrophoresis
3. Polymerase Chain Reaction (PCR)
4. Blotting Methods

7. Restriction enzymes
• Biological or Molecular scissors/Chemical scissors/ that cut double
stranded DNA molecules at specific points.
• Found naturally in a wide variety of prokaryotes
• An important tool for manipulating DNA.
• Restriction enzymes recognize and make a cut within specific
palindromic sequences, known as restriction sites, in the DNA. This is
usually a 4- or 6 base pair sequence.

8. Discovery of RE
• Arbor and Dussoix in 1962 discovered that certain bacteria contain
Endonucleases which have the ability to cleave DNA.
• In 1970 Smith and colleagues purified and characterized the cleavage site
of a Restriction Enzyme.
• Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978
Nobel prize for Medicine and Physiology for their discovery of
Restriction Enzymes.

9. Restriction Endonuclease Types
Type I- multi-subunit, both endonuclease and methylase activities, cleave
at random up to 1000 bp from recognition sequence
Type II- most single subunit, cleave DNA within recognition sequence
Type III- multi-subunit, endonuclease and methylase about 25 bp from
recognition sequence

10. Cleavage
site
Location of
methylase
Examples
Type I Random
Around 1000bp away
from recognition site
Endonuclease and
methylase located on a
single protein molecule
EcoK I
EcoA I
CfrA I
Type II Specific
Within the recognition
site
Endonuclease and
methylase are separate
entities
EcoR I
BamH I
Hind III
Type III Random
24-26 bp away from
recognition site
Endonuclease and
methylase located on a
single protein molecule
EcoP I
Hinf III
EcoP15 I

11. Nomenclature of RE
• The type of bacteria in which the enzyme is found
• The order in which the restriction enzyme was identified and
isolated.
EcoR I is form Escherichia coli bacteria. Strain R .
I as it is was the first E. coli restriction enzyme to be discovered.
Hind III is for Haemophilus influenza
Strain RD (d) and the third (III) endonuclease to be discovered.

12. Picking a palindrome
Words that read the same forwards as backwards
Level leveL
Madam madam
Malyalam malyalaM

13. Palindromes in DNA sequences
Genetic palindromes are
similar to verbal palindromes.
A palindromic sequence in
DNA is one in which the 5’ to
3’ base pair sequence is
identical on both strands.
5
’
5
’
3’
3’

14. blunt end
sticky end

15. “blunt ends” and “sticky ends”
Remember how HaeIII produced a “blunt end”?
EcoRI, for instance, makes a staggered cut and produces a “sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’

16. Some more examples of restriction sites of
restriction enzymes with their cut sites:
HindIII: 5’ AAGCTT 3’
3’ TTCGAA 5’
BamHI: 5’ GGATCC 3’
3’ CCTAGG 5’
AluI: 5’ AGCT 3’
3’ TCGA 5’

17. Cloning Vectors
• Vector is an agent that can carry a DNA fragment into a host cell in
which it is capable of replication.
• If it is used only for reproducing the DNA fragment, it is called a
cloning vector.
• If it is used for expression of foreign gene, it is called an expression
vector.

18. Properties of a good vector
• It should small in size.
• It must have an origin of replication.
• It must also be compatible with the host organism.
• It must possess a restriction site.
• The introduction of donor fragment must not intervene with the self-replicating
property of the cloning vector.
• A selectable marker, possibly an antibiotic resistance gene, must be present to screen
the recombinant cells.
• It should be capable of working under the prokaryotic as well as the eukaryotic system.
• Multiple cloning sites should be present.

19. Types of Vectors
• Plasmid vectors
• Cosmids
• Bacteriophage vectors
• BACs & YACs
• Mini chromosomes

20. Plasmids
• Plasmids were the first vectors to be used in gene cloning.
• They are naturally occurring and autonomously replicating extra-
chromosomal double-stranded circular DNA molecules. However, not
all plasmids are circular in origin.
• They are present in bacteria, archaea, and eukaryotes.
• The size of plasmids ranges from 1.0 kb to 250 kb.
• DNA insert of up to 10 kb can be cloned in the plasmids.

21. • The plasmids have high copy number which is useful for production
of greater yield of recombinant plasmid for subsequent experiments.
• The low copy number plasmids are exploited under certain
conditions like the cloned gene produces the protein which is toxic to
the cells.
• Plasmids only encode those proteins which are essential for their own
replication. These protein-encoding genes are located near the ori.
• Examples: pBR322, pUC18, F plasmid, Col plasmid.

23. pBR322
• It is a circular double stranded DNA and has 4361 base pairs.
• pBR322 also contains the ampR gene (source plasmid RSF2124)
• The tetR gene (source plasmid pSC101), the rop gene encoding a
restrictor of plasmid copy number.
• The plasmid has unique restriction sites for more than 40 restriction
enzymes. • 11 of these 40 sites lie within the tetR gene.
• There are 6 key restriction sites inside the ampR gene

25. PUC 18/19
• It is a circular double stranded DNA and has 2686 base pairs and it
includes:
1. A gene for antibiotic resistance to Ampicillin (ampR).
2. A gene (and its promoter) for the enzyme beta-galactosidase
(lacZ).
3. The lacZ gene contains a polylinker region, with a series of unique
restriction sites called as multiple cloning site (MCS) found in the
plasmid.

27. Advantages of Plasmids in Molecular Biology
• Easy to work with - Plasmids are a convenient size
(generally 1,000-20,000 base pairs).
• Self-replicating - Endless number of copies of the plasmid was obtained
by growing the plasmid in bacteria.
• Stable - Plasmids are stable long-term either as purified DNA or within
bacteria.
• Functional in many species and can useful for
a diverse set of applications - Plasmids can drive gene expression in a
wide variety of organisms, including plants, worms, mice and even cultured
human cells.

28. Major Limitation of Cloning in Plasmids
• Upper limit for clone DNA size is 12 kb
• Requires the preparation of “competent” host cells
• Inefficient for generating genomic libraries as overlapping
regions needed to place in proper sequence
• Preference for smaller clones to be transformed
• If it is an expression vector there are often limitations
regarding eukaryotic protein expression.

29. Bacteriophage Vector
• Bacteriophages or phages are viruses which infect bacterial cells.
• The most common bacteriophages utilized in gene cloning are Phage λ
and M13 Phage.
• A maximum of 53 kb DNA can be packaged into the phage.
• If the vector DNA is too small, it cannot be packaged properly into the
phage.
• Examples: Phage Lambda, M13 Phage, etc.

30. Types of phage vectors
• Insertion vectors- these contain a particular cleavage site where
the foreign DNA of up to 5-11 kb can be inserted.
• Replacement vectors- the cleavage sites flank a region which
contains genes not necessarily important for the host, and these
genes can be deleted and replaced by the DNA insert.

31. Phage Lambda λ
• It has head, tail, and tail fibers.
• Its genome consists of 48.5 kb of DNA and 12 bp ss DNA
which comprise of sticky ends at both the terminals. Since these
ends are complementary, they are cohesive and also referred to as
cos sites.
• Infection by λ phage requires adsorption of tail fibers on the cell
surface, contraction of the tail, and injection of the DNA inside
the cell.

32. Phage Lambda λ

33. M13 Phage
• These vectors are used for obtaining single-stranded copies of the cloned
DNA.
• They are utilized in DNA sequencing and in vitro mutagenesis.
• M13 phages are derived from filamentous bacteriophage M13. The genome
of M13 is 6.4 kb.
• DNA inserts of large sizes can be cloned.
• From the double-stranded inserts, pure single-stranded DNA copies are
obtained.

34. M13 Phage

35. Advantages phase Vectors:
• Useful for cloning large DNA fragments (10 - 23 kbp)
• Inherent size selection for large inserts.
• They are way more efficient than plasmids for cloning large inserts.
• Screening of phage plaques is much easier than identification of
recombinant bacterial colonies.
Disadvantages of phase vectors:
• Less easy to handle

36. • Cosmids - an extra chromosomal circular DNA molecule that combines
features of plasmids and phage; cloning limit - 35-50 kb
• Cosmids are plasmids.
• They are capable of incorporating the bacteriophage λ DNA segment.
This DNA segment contains cohesive terminal sites (cos sites).
• Cos sites are necessary for efficient packaging of DNA into λ phage
particles.
• Large DNA fragments of size varying from 25 to 45 kb can be cloned.
• They are also packaged into λ This permits the foreign DNA fragment or
genes to be introduced into the host organism by the mechanism of
transduction.
Cosmids

37. Cosmid Vector

38. Advantages of Cosmid vector:
• Inherent size selection for large inserts
• Handle like plasmids
• They have high transformation efficiency and are capable of producing a
large number of clones from a small quantity of DNA.
• Also, they can carry up to 45 kb of insert compared to 25 kb carried by
plasmids and λ.
Disadvantages of cosmid vector:
• Not easy to handle very large plasmids
• Cosmids cannot accept more than 50 kb of the insert.

39. GENOMIC LIBRARY
• A genomic library contains all the sequences present in the genome of an organism.
• Genomic library. Gene bank or genomic library is a complete collection of
cloned DNA fragments which comprises the entire genome of an organism
• The difference between both of these libraries is that genomic library comprises
DNA fragments which express the entire genome of an organism while in cDNA
library, mRNA is taken from particular cells of an organism, and then cDNA
consists of this mRNA in a reaction that is catalyzed by an enzyme.

40. • In humans, approximately 25,000 genes exit among the 3 billion base
pairs of DNA in the genome.
• To study anyone of these genes, a researcher first isolates it from
all of the other genes in an organisms DNA.
• One isolation method has a relatively long history and involves the
construction of a DNA library
• When a gene is identified and copied, it is said to have been
“cloned”

41. • The term “library” can refer to a population of organism, each of
which carries a DNA molecule inserted into a cloning vector, or
alternatively to the collection of all of the cloned vector molecules.
• Collection of DNA fragments that have been cloned into vectors so
that researchers can identify and isolate the DNA fragments that
interest them for further study.
• For ease of purification, storage and analysis.

42. Genomic library (made
from genomicDNA)
DNA Library
cDNA Library
(made from cDNA- copyof mRNA)

43. For the construction of Library
• Size of the gene
• Capacity of the vector
• Molecular tools
• Vectors

44. Vectors for Library

45. Steps for construction of genomic library
• Isolation of DNA from cells
• Digestion into small fragments
• Introduction into suitable vectors
• Insertion into bacteria
• DNA isolation
• Collection of Genomic DNA library

46. Summary of Genomic
Library Construction

47. Uses of Genomic Library

48. cDNA bank or cDNA library.
• DNA copy of an mRNA molecule is known as copy
DNA or cDNA (also called complementary DNA).

49. • A cDNA library is a combination of cloned cDNA (complementary DNA)
fragments inserted into a collection of host cells, of which constitute some
portion of the transcriptome of the organism and are stored as a "library".
• cDNA is produced from fully transcribed mRNA found in the nucleus and
therefore contains only the expressed genes of an organism. Similarly, tissue-
specific cDNA libraries can be produced.
• In eukaryotic cells the mature mRNA is already spliced, hence the cDNA
produced lacks introns and can be readily expressed in a bacterial cell. While
information in cDNA libraries is a powerful and useful tool since gene products
are easily identified, the libraries lack information about enhancers, introns, and
other regulatory elements found in a genomic DNA library.

50. Steps in cDNA Library
Construction
• Isolation / Extraction of mRNA
• Construction of cDNA /
complementary DNA

51. Steps in cDNA
Construction.

52. Applications and Uses of cDNA Library
• Storage of reduced amount of information due to the removal of non-
coding regions.
• cDNA can be directly expressed in prokaryotic organisms.
• cDNA libraries are useful in reverse genetics where the additional genomic
information is of less use.
• cDNA library is useful for isolating gene that codes for particular mRNA.

53. Transformation
• Transformation is the direct uptake of exogenous DNA from its
surroundings and taken up through the cell membrane .
• It is the subsequent stable integration & expression of a foreign DNA
into the genome.
• Transformation occurs naturally in some species of bacteria, but it
can also be effected by artificial treatment in other species.
• Cells that have undergone this treatment are said to be competent.

55. Gene Transfer Techniques
• Gene transfer is to transfer a gene from one DNA molecule to another
DNA molecule.
• The directed desirable gene transfer from one organism to another and
the subsequent stable integration & expression of foreign gene into the
genome is referred as genetic transformation.

56. • Transient transformation occur when DNA is not integreted into
host genome
• Stable transformation occur when DNA is integrated into host
genome and is inherited in subsequent generations.
• The transferred gene is known as transgene and the organism that
develop after a successful gene transfer is known as transgenic.

57. Gene Transfer Techniques
• There are mainly 2 methods of gene transfer:
• Indirect (Agrobacterium-mediated) gene transfer
Gene transfer is done by using the bacteria Agrobacterium
tumificiens.
• Direct (Vectorless) gene transfer the gene is directly
transferred into the host by using various techniques.

59. Indirect: Agrobacterium-mediated gene transfer
• Agrobacterium tumefaciens is a soil borne gram negative bacterium.
• It invades many dicot plants when they are injured at the soil level
and causes crown gall disease.
• The ability to cause crown gall disease is associated with the
presence of the Ti (tumour inducing) plasmid within the bacterial
cell.
• Ti plasmid can be used to transport new genes into plant cells.

60. • A remarkable feature of the Ti plasmid is that, after infection, part of the
molecule is integrated into the plant chromosomal DNA .
• This segment, called the T-DNA, is between 15 and 30 kb in size,
depending on the strain.
• T-DNA contains eight or so genes that are expressed in the plant cell and
are responsible for the cancerous properties of the transformed cells.
• These genes also direct synthesis of unusual compounds, called
opines, that the bacteria use as nutrient.
Ti-Plasmid

61. • The vir (virulence) region of the Ti- plasmid contains the
genes required for the T-DNA transfer process.
• The genes in this region encode the DNA processing enzymes
required for excision, transfer and integration of the T-DNA
segment.

62. • The T-DNA region of any Ti
plasmid is defined by the
presence of the right and the left
border sequences.
• These border sequences are 24 bp
imperfect repeats.
• Any DNA between the borders will
be transferred in to the genome of
the plant.

63. • The Ti-Plasmid has an innate ability to transmit bacterial DNA into
plant cells.
• The gene of a donor organism can be introduced into the Ti plasmid
at the T-DNA region
• This plasmid now becomes a recombinant plasmid.
• By Agrobacterium infection, the donor genes can transferred from
the recombinant Ti- Plasmid and integrated into the genotype of the
host plant.
Ti-Plasmid Mediated gene transfer

64. Ti-Plasmid Mediated gene transfer

65. Calcium phosphate mediatedDNA transfer
• The process of transfection involves the admixture of isolated
DNA (10-100ug) with solution of calcium chloride and potassium
phosphate so precipitate of calcium phosphate to be formed.
• Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish.
• A fraction of cells will take up the calcium phosphate DNA
precipitate by endocytosis.

67. PEG mediated transfection
• This method is utilized for protoplast only.
• Polyethylene glycol stimulates endocytosis and therefore DNA
uptake occurs.
• Protoplasts are kept in the solution containing polyethylene
glycol (PEG).
• After transfer of DNA to the protoplast in presence of
PEG and other chemicals, PEG is allowed to get removed

68. • Polyethylene glycol (PEG), in the presence of divalent cations(using
Ca2+), destabilizes the plasma membrane of protoplasts and renders it
permeable to naked DNA.
• In this way, the DNA enters nucleus of the protoplasts and gets
integrated with the genome.
• Culture of protoplasts is taken into a tube and to this tube 40% PEG
4000 (w/v) dissolved in mannitol and calcium nitrate is added slowly.
• Then incubated for few min.

69. Advantages
• A large number of protoplasts can be simultaneously transformed.
• Can successfully use for a wide range of plant species.
Limitations
• The DNA is susceptible for degradation.
• Random integration of foreign DNA into genome may result in undesirable
traits.
• Regeneration of plants from transformed protoplasts is a difficult task.

70. Electroporation
• Electroporation uses electrical pulse to produce transient pores in the
plasma membrane thereby allowing DNA into the cells.
• These pores are known as electropores.
• The cells are placed in a solution containing DNA and subjected to
electrical pulse to cause holes in the membrane.
• The foreign DNA fragments enter through holes into the cytoplasm
and then to nucleus.

72. Advantages of electroporation
• Method is fast.
• Less costly.
• Applied for a number of cell types.
• Simultaneously a large number of cell can be treated.

73. • High percentage of stable transformants can be produced
• Both intact cells and tissue can be transformed.
• The efficiency of transformation depends upon the plant materials
• Disadvantages
• ~40 to 50% incubated cells receive DNA
• ~50% of the transformed cells can survive

74. Gene gun Method or Biolistics or Microprojectiles
or biolistic particle delivery system
• Biolistics or particle bombardment is a physical method that uses
accelerated microprojectiles to deliver DNA or other molecules into
intact tissues and cells.
• The gene gun is a device that literally fires DNA into target cells .
• The DNA to be transformed into the cells is coated onto
microscopic beads made of either gold or tungsten.

75. • The coated beads are then attached to the end of the plastic bullet and
loaded into the firing chamber of the gene gun.
• An explosive force fires the bullet with DNA coated beads towards the
target cells that lie just beyond the end of the barrel.
• Some of the beads pass through the cell wall into the
cytoplasm of the target cells

77. • Advantages
• This method can be use to transform all plant species.
• Transformation protocol is relatively simple.
• Disadvantages
• High cost of the equipment and microcarriers.
• Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.).
• Transfer DNA is not protected.

78. Polymerase Chain Reaction (PCR)
• PCR is a technique that takes specific sequence of DNA of small amount
and amplifies it to be used for further testing.
• PCR is In vitro technique
• In 1983 Dr. Kary Mullis developed PCR
• To amplify a lot of double-stranded DNA molecules (fragments) with same
(identical) size and sequence by enzymatic method and cycling condition.

79. Basic requirements for PCR reaction

80. Steps in PCR
1. Denaturation of ds DNA template
2. Annealing of primers
3. Extension of ds DNA molecules

81. Denaturation
Single stranded
92C
3’
• Temperature: 92-94C
• Double stranded DNA melts
DNA
5’
3’ 5’
+
5’3’
5’ 3’

82. Annealing
• Temperature: ~50-70C (dependant on the melting
temperature of the expected duplex)
• Primers bind to their complementary sequences
5’3’
5’ 3’
Forward primer Reverse primer

83. Extension
• Temperature: ~72C
• Time: 0.5 - 3min
• DNA polymerase binds to the annealed primers and
extends DNA at the 3’ end of the chain
Taq
5’
3’
Taq5’

84. Cycling

85. Products of Extension
3’5’
3’ 5’
3’5’
3’ 5’
Taq
Taq

86. Overall Principle of PCR
• DNA – 1 copy
•
Known sequence Sequence of interest
• PCR

87. Advantages of PCR
• Small amount of DNA is required per test
• Result obtained more quickly - usually within 1 day for PCR
• Usually not necessary to use radioactive material (32P) for
PCR.
• PCR is much more precise in determining the sizes of alleles -
essential for some disorders.
• PCR can be used to detect point mutations.

89. Types of polymerase chain reaction-PCR
• Real-Time PCR (quantitative PCR or qPCR)
• Reverse-Transcriptase (RT-PCR)
• Multiplex PCR
• Nested PCR
• High Fidelity PCR
• Fast PCR
• Hot Start PCR
• GC-Rich PCR
• Long-range PCR
• Arbitrary Primed PCR

90. PCR Machine

91. THANK
YOU