rDNA for MBBS students

1. RECOMBINANT
DNA
TECHNOLOGY

2. BIOTECHNOLOGY ?
INTEGRATING BIOLOGY AND TECHNOLOGY
EXPLOITING FOR INDUSTRIAL OR OTHER
PURPOSES
GENETIC MANIPULATIONS INCLUDED

3. RECOMBINANT DNA
TECHNOLOGY
Manipulating genetic material
to achieve our goals.

4. HISTORY
BOYER & COHEN – 1973
Recombined pSC 101 and pSC
102 and cloned the new plasmid
in E.coli

5. Human genome-
23 pairs of chromosome
109 base pairs ( 3 billion)
20,000-30,000 proteins
coding genes
3 inventions made it
possible
Diagnosis of genetic disease
Gene therapy- partial
success

6. ASPECTS OF R-DNA TECHNOLOGY
1. MOLECULAR TOOLS
2. HOST CELLS
3. VECTORS
4. METHODS OF GENE TRANSFER
5. GENE CLONING STRATEGIES

7. MOLECULAR TOOLS
ENZYMES
LYSING CLEAVING SYNTHESIZING JOINING OTHER

8. CLEAVING ENZYME
EXONUCLEASE ENDONUCLEASE

9. RESTRICTION ENDONUCLEASES
• Bacterial enzymes
• Cut at specific sites
• Restriction sites
• Discovery? Haemophilus influenzae
• Isolation? E coli
• Arber ,Smith and Nathan- nobel prize for
discovery

10. RESTRICTION ENDONUCLEASES
• Identify 4-8 base pairs (bp)
• Palindromic sequence
• Restrict expression of non bacterial DNA

11. Nomenclature
• First letter - bacterium’s genus from which
isolated
• Next two letters- bacterium’s species
• Fourth letter- strain
• Numeral- order of isolation

12. Escherichia coli RY13 – Eco RI
Bacillus amyloliquefaciens H – Bam HI
Escherichia coli R245 – Eco RII

13. RESTRICTION
SITE
• 4-8 bp long
• Palindromic

14. TYPES
• I - not specific, bifunctional, cuts away from
target sequence, Mg
• II - very specific, cuts adjacent to sequence,
Mg
• III – very specific , bifunctional, cuts 25 bp
away from sequence, ATP

15. TYPES OF ENDS PRODUCED POST
CUTTING
BLUNT STICKY

16. Blunt – No H bonding on rejoining
Sticky – H bonding on rejoining
Sticky Ends are more used in rDNA
Both can be joined by ligase

18. LYSING ENZYMES
• LYZOZYME
• CELLULASE
• PECTINASE
• CHITINASE
• PROTEASE
• LIPASE

19. SYNTHESIZING ENZYMES
• DNA polymerase
• Reverse transcriptase

20. DNA LIGASES
• Join cut pieces of DNA
• Found in – Viruses / E.coli / Eukaryotes
• Forms phosphodiester bond between
fragments ( 5’ & 3’) since hydrogen bond not
strong enough

21. OTHERS
• Alkaline phosphatase- prevents circling of DNA
post cutting

22. HOST CELLS (Cloning Hub)
• Living system in which vector can be
propagated
• Should multiply easily
• Hence, microorganisms preferred

23. Prokaryotic Hosts
Mostly E.coli
Drawback – no post-translational modifications
Eukaryotic Hosts
Preferred for human proteins
Mostly, yeast
Sometimes, mammalian cells (mouse) – post
translational modifications done

24. VECTORS ( Cloning vehicles)
• Carries/transfers DNA of interest to be cloned
• Self replicating
• Needs ori
selectable marker gene
cloning site

25. Plasmid

26. PLASMID
• Most common
• Double stranded, circular
• In bacteria
• Autonomous replication
• Extra-chromosomal
• pBR322 , pBR327 , pUC18 , Ti plasmid
• O.5-8 kbp

27. BACTERIOPHAGES
• phage
• M 13 phage
• 9-23 kbp
• Virus that replicates in bacteria
Carries larger DNA segment than plasmid, Can
inject DNA into bacterium with more efficiency
than Plasmid
VIRUSES
• CMV, Adenovirus, retrovirus

28. COSMID
• Artificial virus
• Bacteriophage+ plasmid
• Cos site- 12 bp ( Packaging of DNA into phage)
• 30 – 45 kbp

29. ARTIFICIAL CHROMOSOMES
• BAC 50 – 300 kbp
• YAC 1000-2500 kbp
• HAC

30. PASSENGER DNA
• Desired gene
• Types –
1. cDNA – DNA from RNA
2. Synthetic dna – whole gene artificial
3. Random DNA – gene directly from cell by
using enzymes

31. PROCESSES
1. Isolation
2. Cutting
3. Amplification
4. Formation of r DNA
5. Transfer into host cells
6. Selection and screening
7. Obtaining desired product

32. Cloning

33. ISOLATION
• Culture of bacteria grown
• Cells broken up
• Extract is treated ( protease,
RNAse)
• DNA ppt out after adding chilled
• ethanol

34. CUTTING AT SPECIFIC SITES
• Using restriction enzymes
• Electrophoresis to check progress
fragments separated
smaller moves farther
-ve molecules move towards anode
results seen under UV after staining with
ethidium bromide
needed fragments cut out ( elution)

36. AMPLIFICATION
• By PCR – polymerase chain reaction
• Production and amplifying billions of copies of
fragment of DNA
• Invented by Kary Mullis

37. TRANSFER INTO HOST CELLS
• Transformation – into bacterial cells
DNA hydrophilic
cold shock(CaCl2) and heat shock
(0-5 C) (37-45 C)
• Conjugation – natural process
donor and recipient join by
cytoplasmic bridges

38. • Electroporation – simple and rapid. Cellular
uptake of exogenous DNA. Electric field
mediated permeabilization
• Liposomes – lipid molecules with aqueous
interior. Lipofection.
• Microinjection & particle bombardment –
direct transfer

39. SELECTION AND SCREENING
• Recombination – chance event
• One in 1000s
• Types seen – non transformants
transformants with vector
transformants with r dna

40. OBTAINING PRODUCT
• Host cells will produce the protein the gene
encodes
• Cultured at large scale
• Levels – lab level
pilot plant level – bioreactors (batch
and continuous cultures)
industrial level – downstream processing

42. POLYMERASE CHAIN REACTION
• In vitro cell free amplification technique
• Specific DNA taken
• KARY MULLIS – 1984
• REQUIREMENTS :
1. Target DNA
2. Two primers
3. d ATP / d CTP / d GTP / d TTP
4. DNA polymerase – Taq heat resistant, fresh
addition not required.

43. PRINCIPLE OF PCR
1. Denaturation: dsDNA 2 separate
strands.
2. Renaturation/Annealing : each strand hybridizes
with a primer attaching to complementary
region at 55*C, high primer conc.(1min)
3. Synthesis: begins at 3’OH end of each primer,
extended by joining complementary bases to
DNA strands.(taq polymerase, 2min,75*C,95*C
stop)
4. Total time for 1cycle = 3 -5min
95*C, 1min

47. VARIATIONS OF PCR
• Nested
• Inverse
• Anchored
• Reverse transcriptase
• Asymmetric
• Real time quantitative
• Random amplified polymorphic DNA (RAPD)
• Amplified fragment length polymorphism (AFLP)
• Rapid amplification of c DNA (RACE)

48. APPLICATIONS OF PCR
• Prenatal diagnosis of inherited disease
(Sickle cell anemia, PKU, Thalassemia)
• Criminal identification
• Diagnosis of – cancer (Cervical-HPV)
retroviral infection (HIV)
bacterial infection (TB)
• Sex determination

50. APPLICATIONS of rDNA
• MOLECULAR BASIS OF DISEASES – sickle cell,
dystrophy, cystic fibrosis
• DIAGNOSIS OF DISEASES – existing diseases,
response to drugs
• PRENATAL DIAGNOSIS

51. PROTEIN FUNCTION
Anti coagulant TPA for heart attacks
Growth hormone For dwarfism
Erythropoietin For anemia
Blood factors Hemophilic patients
Insulin For diabetes
Vaccines Prevention
Interferon Cancer treatment
interleukins HIV /cancer/ immunodeficiency

52. • GENE THERAPY - normal genes replaces defective.
ULTIMATE cure for genetic diseases.
• GERM LINE GENE THERAPY – germ cells modified then
combined. Heritable changes.
• SOMATIC GENE THERAPY – genes into somatic cells. Not
heritable.
• EX VIVO GENE THERAPY – in cultured cells
(bone marrow) which are then transferred.
• IN VIVO GENE THERAPY – direct delivery into particular
tissue’s cells.

55. • SCREENING TESTS – for genetic diseases.
Performed on parents. Sometimes, foetus too.
In utero treatment can be given.
• TRANSGENIC ANIMALS – DNA of other species
introduced in host animal. Transgenes passed
on to offsprings.
1. A model for understanding diseases.

56. • MICE – used because a mammal.
used to study cancer, dystrophy,
alzeihmer’s, arthritis

57. • PRODUCING PROTEINS – by ‘pharm’ animals
Cow – lactoferrins, interferons
Goat – TPA
Mouse – immunoglobulins
Pig - Haemoglobin
• VACCINES - k/a new generation of vaccines
high yield, no allergy, effective
TB, Tetanus, Typhoid

59. TECHNIQUES FOR IDENTIFICATION
OF DESIRED MOLECULE
• NORTHERN BLOTTING – RNA
• SOUTHERN BLOTTING – DNA
• WESTERN BLOTTING - Proteins

60. Southern
Blotting

61. DNA FINGERPRINTING
• DNA Fingerprinting is a laboratory technique
used to establish a link between biological
evidence and a suspect in a criminal
investigation.
• A DNA sample taken from a crime scene is
compared with a DNA profiles are a match,
then the evidence came from that suspect

62. DNA LIBRARY
• Collection of cloned restriction fragments of
the DNA of an organism
• TYPES –
1. GENOMIC DNA – introns + exons
2. COMPLEMENTARY DNA – only exons
specialized

63. cDNA

65. Probes
• Cleavage of large DNA molecules by restriction
enzymes produces a bewildering array of fragments.
How can the DNA sequence of interest be picked out of
a mixture of thousands or even millions of irrelevant
DNA fragments?
• The answer lies in the use of a probe—a short piece of
ssDNA, labeled with a radioisotope, such as 32P, or
with a nonradioactive molecule, such as biotin.
• The sequence of a probe is complementary to a
sequence in the DNA of interest, called the target DNA.
Probes are used to identify which band on a gel or
which clone in a library contains the target DNA, a
process called screening.

66. Probe in diagnosis of sickle cell disease

67. Allele specific probes

69. Microarray

70. Cloned DNA
sequencing

71. RFLP
• Restriction Fragment Length Polymorphism
• Its a genetic variant that can be observed by
cleaving the DNA into fragments (restriction
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, for example, in prospective parents or
in fetal tissue.

72. • DNA genome has difference of about 0.1% among
individuals
• 1 base pair change in 1200
• Change can lead to mutation or polymorphism
• polymorphism is a clinically harmless DNA
variation that does not affect the phenotype.
• It is traditionally defined as a sequence variation
at a given locus (allele) in more than 1% of a
population.
• Polymorphisms primarily occur in regions of the
genome that do not encode proteins, that is, in
introns and intergenic regions.

73. DNA variations in RFLP

74. Single base changes in DNA
• 90% of human genome variation comes in the form of
single-nucleotide polymorphisms, (SNPs, pronounced
“snips”)
• The 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 by the same
mechanism. In either case, cleavage with an endonuclease
results in fragments of lengths differing from the normal,
which can be detected by DNA hybridization
• The altered restriction site can be either at the site of a
disease-causing mutation (rare) or at a site some distance
from the mutation

75. Tandem Repeats
• Alternatively, polymorphism in chromosomal DNA can arise from the
presence of a variable number of tandem repeats (VNTR)
• These are short sequences 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,
there- fore, serves as a molecular fingerprint.
• Cleavage by restriction enzymes yields fragments that vary in length
depending on how many repeated segments are contained in the
fragment. Variation in the number of tandem repeats can lead to
polymorphisms.
• Many different VNTR loci have been identified, and are extremely useful
for DNA fingerprint analysis, such as in forensic and paternity identity
cases.
• It is important to emphasize that these polymorphisms, whether SNP or
VNTR, are simply markers, which, in most cases, have no known effect on
the structure, function, or rate of production of any particular protein.
Microsatellites are a type of VNTR.(Seen around centromere and
telomeres)

77. Prenatal diagnosis
• DNA sample – Amniotic
fluid, Chorionic villus
• AFP measurement in
sample
• Karyotyping
• USG for visual
anatomical defect such
as Neural tube defects

78. Sickle cell diagnosis

79. THANK YOU

80. Reference
• Textbook of Biochemistry, Lippincott