its about genetic engineering.

1. Genetic Engineering
Dr. Pankaj KambleDr. Pankaj Kamble

2. Contents
 Introduction
 Basic principle of recombinant DNA technology
 Molecular tools of genetic engineering
 Methods
 Applications recombinant DNA technology
 Gene library
 Blotting techniques
 RFLP
 PCR

3. Introduction
Genetic recombination technology consists of
the breakage and joining of DNA molecules.
Genetically engineered DNA prepared by transplanting
or splicing genes from one species into the cells of a
host organism of a different species. Such DNA
becomes part of the host's genetic makeup and is
replicated.
Genetic engineering primarily involves the
manipulation of genetic material ( DNA) to achieve
the desire goal in pre determined way.

4. • Other terms – Recombinant DNA technology
Gene manipulation
Gene cloning
Genetic modifications
New genetics

5. Basic principle of recombinant
DNA technology
• Manipulation and alteration of genes
Artificially copying a piece of DNA from one
organism and joining this copy of DNA into the
DNA of another organism

6. Basic principle of rDNA technology

8. Molecular tools of genetic engineering
• The genetic engineer's tool kit or molecular tool
namely the enzymesenzymes are most commonly used in
recombinant DNA experiments are
 Restriction endonucleases -DNA cutting Enzyme.
 DNA Ligases- DNA joining Enzyme.

9. Restriction enzymes act as molecular
scissors and cut DNA at specific sites called
restriction sites
Restriction endonucleases
Restriction site
Restriction
site Restriction
ezymes

10. • Named with particular reference to the
bacteria from which they are isolated.
Eg. EcoR‫ו‬
:Types 3
Restriction endonucleases

15. DNA ligase

16. Host cells: the factor of cloning
The hosts are the living system or cell in
which the carrier of recombinant DNA
molecule or vector can be propagated.
Prokaryotic
Bacteria Escherichia coli
Bacillus subtilis
Streptomyces sp

17. Eukaryotic
1.Fungi Saccharomyces cerevisiae
Aspergillus nidulans
2.animals Insect cells
Oocytes
Mammalian cells
Whole organisms
3.plants Protoplast
Intact cell
Whole plants

18. Vectors are the DNA molecule, which can carry
A foreign DNA fragment to be cloned. The are
self replicating in an appropriate host cell.
 The most important vectors are Plasmids,
Bacteriophages, cosmids.
An ideal characteristics of an vector is should
be small in size with single endonuclease site.
But natural occurring rarely posses this
characteristics.
Cloning vectors

19. Plasmids

21. Bacteriophage λ (Lambda)

22. Cosmid
•Cosmids are vectors posses the characteristic of
both plasmid and bacteriophage.
•Cosmids can be constructed by adding a fragment
of phage DNA to plasmid.
•A foreign DNA 40 Kb can be inserted into cosmid
DNA

23. BACs: Bacterial Artificial Chromosomes
•The construction BAC is based on F
plasmid which is large than other plasmid
used as cloning vector.
•BACs can accept DNA inserts of around
300kb

26. YACs: Yeast ArtificialYACs: Yeast Artificial
ChromosomesChromosomes
Yeast Artificial Chromosomes (YAC)Yeast Artificial Chromosomes (YAC) is a synthetic DNA
that can accept large fragment (particular human DNA).
It is possible to clone large DNA pieces by using YAC.

27. Human Artificial Chromosomes
• Synthetically produced vector DNA,
possessing the characteristic of human
chromosome
• Size range from 1/10th
to 1/5th
of human
chromosome

29. Methods of gene Transfer
Transformation
Electroporation
Conjugation
Transduction
Microinjection
Liposome-mediated gene transfer.

30. Transformation

31. Electroporation
• Application of
high voltage
electrical field
to cells

33. Liposome mediated gene transfer

34. Direct transfer
• Microinjection
• Particle bombardment

35. Applications of Recombinant DNA
Technology
1)Large-scale production of human proteins by
genetically engineered bacteria.
•Recombinant human insulin
•Recombinant human growth hormone
•Recombinant blood clotting factors (VIII, IX,
tPA)
•Recombinant hepatitis B vaccine, HPV vaccine
•Cytokines and growth factors (IF, IL)
•Monoclonal antibodies
•Recombinant enzymes
•Recombinant HIV protein for ELISA testing
•Albumin, fibrinolytic and thrombolytic agents

36. Applications of Recombinant DNA
Technology
2) Gene therapy for genetic diseases
3) Food production
4) Plant: genetically modified corn

37. Gene library
• A DNA library is a collection of cloned
restriction fragments of the DNA of
an organism
• 2 types
1. Genomic library
2. cDNA library

38. Genomic DNA libraries
• Collection of DNA fragments from a
particular species.
• Constructed by isolating the entire DNA
from a cell which is cut into fragments and
cloned in suitable vector.

39. Gene library for humans
• Each human chromosome, containing
approximately 100000 kb can be cut
into about 25000 DNA fragments of
average size of 4 kb.
• As we have 23 different chromosomes,
there are total of 575000 fragments of
4 kb formed.

40. cDNA library
• cDNA libraries contain those DNA
sequences that appear as mRNA
molecules, and these differ from one
cell type to another.
• This mRNA can be used as a template to
make a complementary dsDNA (cDNA)
molecule using the enzyme reverse
transcriptase.

42. BLOTTING TECHNIQUES
• Blots are techniques for transferring DNA ,
RNA and proteins onto a carrier so they
can be separated, and often follows the
use of a gel electrophoresis.
• The Southern blot is used for transferring
DNA, the Northern blot for RNA and the
western blot for PROTEIN.

43. Southern blotting
• Named after scientist Edward Southern
who developed it in 1975
• Other names are laboratory jargons
which are now accepted eg. Northern
and western blotting

44. Southern blotting

45. Southern blotting

46. Southern blotting

47. Northern Blotting

49. Restriction Fragment Length
Polymorphism (RFLP)

50. Restriction Fragment Length
Polymorphism (RFLPs)
• A polymorphism is a clinically harmless
DNA variation that does not affect the
phenotype.
• At the molecular level, polymorphism is a
variation in nucleotide sequence from one
individual to another.
• Polymorphisms often occur in the
intervening sequences that do not code
for proteins (Introns).

51. Restriction Fragment Length
Polymorphism (RFLPs)
• RFLP is a genetic variant that can be examined
by cleaving the DNA into fragments with a
restriction enzyme.
• The length of the restriction fragments is
altered if the genetic variant alters the DNA so
as to create or abolish a site of restriction
endonuclease cleavage (a restriction site).
• RFLP can be used to detect human genetic
variations.

52. Polymorphism vs Mutation
• A polymorphism is a clinically harmless DNA
variation that does not affect the phenotype.
• In contrast, mutation refers to an infrequent, but
potentially harmful, genome variation that is
associated with a specific human disease.
• Polymorphism: sequence variation at a given
locus in more than 1% of population.

53. R3R2R1
R1 R3
DNA1
DNA2
Restriction
endonuclease
Restriction
endonuclease
4 fragments
3fragments
Restriction Fragment Length Polymorphism (RFLP)

54. DNA variations resulting in RFLP
Single nucleotide polymorphism(SNP)
• About 90% of human genome variation comes
in the form of SNP.
• Variations that involve just one base
• Substitution of one nucleotide at a
restriction site can render the site
unrecognizable by a particular restriction
endonuclease.
• A new restriction site can also be created.

56. DNA variations resulting in RFLP
Variable Number of Tandem Repeats (VNTR)
• Short sequences (10-100 bp) of DNA at
scattered locations in the genome, repeated in
tandem (one after another).
• The number of these repeat units varies from
person to person, but is unique for any given
individual and, therefore, serves as a molecular
fingerprint.

58. APPLICATIONS
1.Diagnosis of genetic defects
2.Study of genetic individuality
3.Detection of changes in DNA
sequences
4.Linkage studies

59. Direct diagnosis of sickle cell disease using RFLP

61. Polymerase Chain ReactionPolymerase Chain Reaction

62. Polymerase Chain Reaction (PCR)
PCR is an in vitro technique for the amplification of a
region of DNA.
Cell free amplification technique.
Developed by Kary Mullis in the 1980s

63. Principle
Double stranded DNA of interest is denatured to separate
strands.
Each strand is then allowed to hybridize with a primer.
The primer template duplex is used for synthesis.
This three steps:Denaturation,anneling and extension repeated
again and again to generate multiple forms of target DNA.
The primer extension product synthesized in 1 cycle serve as
template for next cycle.

64. TEACHNIQUE OF PCR
 Target DNA (100-35,000 bp IN LENGTH).
 Two primers(these are typically short, single
stranded oligonucleotides which are
complementary to the outer regions of known
sequence.
 Buffer(tris buffer ,KCl & MgCl2)
 4 Deoxyribonucleotide(dATP, dCTP, dGTP, dTTP)
 A DNA polymerase that can withstand the
temperature upto 95o
C.

65. PCR tubes are manufactured from polypropylene.
Ultra thin wall design for efficient heat transfer.
Compatible with standard 96-well heat blocks.
Autoclavable.
DNase and RNase free.

66. PCR ProtocolPCR Protocol
•Mix DNA, primers, dNTPs, Taq,
buffer, Mg2+
•Program thermocycler for times
and temps
–denaturation
–annealing
–Extension
•20-40 cycles
It helps to keep the enzymes such as Taq Polymerase in their
optimal confirmation.

67. Mix DNA, primers, dNTPs,
Taq, buffer, Mg2
Eppendorf’s tube

68. Laboratory apparatus used to amplify segments of DNA via the
polymerase chain reaction (PCR) process.
Thermal cycler
PCR consists of a series of 20-40 repeated temperature changes,
called cycles, with each cycle commonly consisting of 2-3 discrete
temperature steps.
PCR consists of a series of 20-40 repeated temperature changes,
called cycles, with each cycle commonly consisting of 2-3 discrete
temperature steps.

69. •:
Denaturation at 94°C which lasts for 1 min
Annealing at 54°C which lasts for 1 min
extension at 72°C which lasts for 2 min

70. 1 cycle = 2 copies

71. 2 cycle = 4 copies

72. 3 cycle = 8 copies

73. 4 cycle = 16 copies

74. 5 cycle = 32 copies

75. 6 cycle = 64 copies

76. 7 cycle = 128 copies

77. 8 cycle = 256 copies

78. TYPES OF PCR
• REAL-TIME PCR
• NESTED PCR
• INVERSE PCR
• REVERSE TRANSCRIPTION PCR
• ASYMMETRIC PCR
• MULTIPLEX PCR

79. Applications of PCR
1. Diagnosis:
Bacterial and viral diseases eg., TB, Hepatitis
C, CMV, HIV
2. Medicolegal cases: DNA amplification from
hair follicles and blood sample followed by
RFLP
3. Diagnosis of genetic disorders: SCD,
thalassemia, cystic fibrosis
4. Prenatal diagnosis of inherited disorders

80. Applications of PCR contd…
5. Cancer detection:
i. To monitor residual abnormal cells present
in treated patients.
ii. Identification of mutation in
oncosuppressor genes eg., p53
6. Fossil studies: to study evolution by comparing
the sequences in the extinct and living
organisms

81. DNA
FINGERPRINTING

82. DNA FINGERPRINTING
• Technique employed by forensic scientists to
assist in identification of individuals by their
respective DNA profiles.
• DNA of every individual is unique; chemical
structure being same in everyone, difference
lies in order of base pairs
• Developed by Prof. Alec Jeffreys.

83. • This method uses repetitive DNA sequences
that are highly variable called variable no. of
tandem repeats (VNTRs).
• VNTR loci are very similar in closely related
humans like parent and child and identical in
monozygotic twins but they are so variable that
unrelated individuals are extremely unlikely to
have the same VNTRs.
DNA FINGERPRINTING Contd…

85. APPLICATIONS
•Paternity-maternity disputes – father
identification cases, confirming legal nationality,
and instances of adoption.
•Criminal identification cases - genetic evidences
left at crime spot can be compared with the
VNTR pattern of crime suspect.
•Personal identification – children separated from
parents during floods, tsunami etc. may be united
with their parents with the help of DNA
fingerprinting.
•Creating population data banks – one of the best
way to keep identification record of a person.

86. THANK YOU