Recombinant dna technology

1. Recombinant DNA Recombinant DNA
TechnologyTechnology
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. A – A good student is liked by teacher
G – Greets everyone with smile
O – Obedient
O – On time for college
D – Dresses neatly
S – Studies with interest
T – Treats everyone with smile
U – Understands everything
D – Does daily home work
E – Eager to know new things
N – Never misbehaves
T – Talks little in class

3. Biotechnology may be defined as “the
method by which a living organism or its
parts are used to change or to incorporate a
particular character to another living
organism”
It involves the application of scientific
principles to the processing of materials by
biological agents.

4. Genetic recombination is the exchange of
information between two DNA segments.
This is a common occurrence within the same
species.
But by artificial means, when a gene of one
species in transferred to another living organism,
it is called recombinant DNA technology.
In common, this is known as genetic
engineering.

5. Definition of Definition of
recombinant DNArecombinant DNA
•Production of a unique DNA molecule by
joining together two or more DNA
fragments not normally associated with
each other
•DNA fragments are usually derived from
different biological sources

6. Definition of recombinant Definition of recombinant
DNA technologyDNA technology
•A series of procedures used to recombine
DNA segments. Under certain conditions, a
recombinant DNA molecule can enter a cell
and replicate.

7. History of recombinant History of recombinant
DNA technologyDNA technology
•Recombinant DNA technology is one
of the recent advances in
biotechnology, which was developed
by two scientists named Boyer and
Cohen in 1973.

8. Development of Development of
molecular biologymolecular biology
•Early research on prokaryotic genetics and
the development of molecular techniques has
led to a new discipline called MOLECULAR
BIOLOGY
•“Tools” have been developed (and still
continue to be modified/improved) to enable
scientists to examine very specific regions of
the genome or genes.

9. •Advances in Molecular Biology
–The combination of
restriction/modification enzymes and
hybridization techniques enable the
application of a wide variety of
procedures

10. TECHNIQUES and PROCEDURES
•Gene isolation/purification/synthesis
•Sequencing/Genomics/Proteomics
•Polymerase chain reaction (PCR)
•Mutagenesis (reverse genetics)
•Expression analyses (transcriptional and translational
levels)
•Restriction fragment length polymorphisms (RFLPs)
•Biochemistry/ Molecular modeling
•Gene therapy

11. Applications
•Quantitative preparation of biomolecules
•Recombinant Vaccines
•Antenatal diagnosis of genetic diseases
•Monoclonal antibodies
•Cell/tissue culture
•To identify mutations in genes
• Xenotransplantation

12. Contd…Contd…
•To detect activation of oncogenes
•Production of next generation
antibiotics
•Forensics
•Biosensors
•Genetically modified crops
• Bioterrorism detection

13. 1.Quantitative Preparation of 1.Quantitative Preparation of
BiomoleculesBiomolecules
•If molecules are isolated from higher
organisms, the availability will be greatly
limited.
•For eg.- To get 1 unit of growth hormone,
more than 1000 pituitaries from cadavers
are required.
•By means of recombinant technology, large
scale availability is now assured.

14. 2. Risk of contamination is Eliminated2. Risk of contamination is Eliminated
•It is now possible to produce a biological
substance without any contamination.
•Hepatitis, caused by HBV, is highly contagious.
•Preparations of vaccines or clotting factors are
free from contaminants such as hepatitis B
particles.
•RD-Technology provides the answer to produce
safe antigens for vaccine production.

15. 3. Specific probes for Diagnosis of 3. Specific probes for Diagnosis of
DiseasesDiseases
Specific probes are useful for:
i. Antenatal diagnosis of genetic diseases.
For eg.- many of the single gene defects
like cystic fibrosis, phenyl ketonuria etc.
Could be identified by taking cell samples
from fetus.
ii. To identify viral particles or bacterial DNA
in suspected blood and tissue samples.

16. Contd…Contd…
iii. To demonstrate virus integration in
transformed cells.
iv. To detect activation of oncogenes in
cancer.
v. To pinpoint the location of a gene in a
chromosome.
vi. To identify mutations in genes.

17. 4. Gene Therapy4. Gene Therapy
•It is an important applications of RD-Technology
• Normal genes could be introduced into the patient so
that genetic diseases can be cured.

18. •How to find one gene in large genome?
•A gene might be 1/1,000,000 of the genome.
Three basic approaches:
•1. Cell-based molecular cloning: create and
isolate a bacterial strain that replicates a copy of
your gene.
•2. Polymerase chain reaction (PCR). Make
many copies of a specific region of the DNA.
•3. Hybridization: make DNA single stranded,
allow double strands to re-form using a labeled
(e.g. radioactive) version of your gene to make it
easy to detect.

19. Basic principle of recombinant Basic principle of recombinant
DNA technologyDNA technology
•The DNA is inserted into another DNA
molecule called ‘vector’
•The recombinant vector is then
introduced into a host cell where it
replicates itself, the gene is then
produced

21. Cell-Based Molecular Cell-Based Molecular
CloningCloning
•The original recombinant DNA technique: 1974 by
Cohen and Boyer.
•
•Several key players:
• 1. restriction enzymes. Cut DNA at specific
sequences. e.g. EcoR1 cuts at GAATTC and BamH1
cuts at GGATCC.
•
–Used by bacteria to destroy invading DNA: their own
DNA has been modified (methylated) at the
corresponding sequences by a methylase.
•

22. 2. Plasmids: independently replicating DNA circles (only
circles replicate in bacteria). Foreign DNA can be
inserted into a plasmid and replicated.

–Plasmids for cloning carry drug resistance genes
that are used for selection.
–Spread antibiotic resistance genes between bacterial
species
3. DNA ligase. Attaches 2 pieces of DNA together.

4. transformation: DNA manipulated in vitro can be put
back into the living cells by a simple process .
–The transformed DNA replicates and expresses its
genes.

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25. II. Restriction EndonucleasesII. Restriction Endonucleases

26. A. Origin and functionA. Origin and function
•Bacterial origin = enzymes that cleave foreign
DNA
•Named after the organism from which they
were derived
–EcoRI from Escherichia coli
–BamHI from Bacillus amyloliquefaciens
•Protect bacteria from bacteriophage infection
–Restricts viral replication
•Bacterium protects it’s own DNA by
methylating those specific sequence.

27. B. AvailabilityB. Availability
•Over 200 enzymes identified, many
available commercially from
biotechnology companies

28. C. ClassesC. Classes
•Type I
–Cuts the DNA on both strands but at a non-
specific location at varying distances from
the particular sequence that is recognized
by the restriction enzyme
–Therefore random/imprecise cuts
–Not very useful for rDNA applications

29. •Type II
–Cuts both strands of DNA within the
particular sequence recognized by the
restriction enzyme
–Used widely for molecular biology
procedures
–DNA sequence = symmetrical

30. •Reads the same in the 5’ 3’ direction on
both strands = Palindromic Sequence
•Some enzymes generate “blunt ends” (cut in
middle)
•Others generate “sticky ends” (staggered
cuts)
–H-bonding possible with complementary tails
–DNA ligase covalently links the two fragments
together by forming phosphodiester bonds of the
phosphate-sugar backbones

32. DNA Ligase in Action!DNA Ligase in Action!

33. III. Vectors for Gene III. Vectors for Gene
CloningCloning

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