Recombinant DNA Technology for MBBS course

1. Recombinant DNA Technology
Dr. Farhana Atia
Assistant Professor
Department of Biochemistry
Nilphamari Medical College, Nilphamari
Email: farhana.atia@gmail.com

2. RED BIOTECHNOLOGY WHITE BIOTECHNOLOGY
GREEN
BIOTECHNOLOGY
BLUE
BIOTECHNOLOGY
Medical biotechnology: manufacture
pharmaceuticals, used in molecular
diagnostic.
Industrial biotechnology: applied to
industrial and other production
processes.
Agricultural biotechnology: applied to
agricultural processes and products.
Marine biotechnology: marine and
aquatic applications of
biotechnology
BIOTECHNOLOGY

3. Medical Biotechnology
Medical Biotechnology is the use of living cells and
cell materials to research and produce
pharmaceutical and diagnostic products that help
to treat and prevent human diseases.
Applications
1.Pharmacology: Insulin, GH, antibiotic, clotting
factor, antibody production
2.Gene therapy: Somatic/ germ line
3.Stem cells
4.Tissue engineering

4. • Stem cell therapy: Bone marrow transplants,
replacing damaged heart tissue after a heart
attack and replacing damaged nerve tissue in
spinal cord injury.
• A form of regenerative medicine, tissue
engineering is the creation of human tissue
outside the body for later replacement.

5. Recombinant DNA technology
• Manipulation of genetic material (DNA) in the
laboratory involving
– isolation & end to end joining of DNA sequences
– from very different sources
– to make chimeric molecules
are collectively referred to as rDNA technology.
• Other terms are
Gene manipulation Genetic engineering
Gene cloning Genetic modifications

6. Tools & techniques involved in rDNA technology
• Restriction enzymes (DNA
cutting)
• DNA ligase (joining)
• Cloning (DNA amplification)
– Host cell (factories)
– Vector (vehicle)
• Gene transfer method
(Transformation,
Conjugation, direct transfer,
electroporation)
• Library (collection
of clones)
• Probes
• Isolation &
purification of NA
• Blotting &
hybridization
• DNA sequencing
• PCR

7. Restriction enzyme
• Endonuclease
– Cut DNA at specific DNA sequences within the
molecule
– Into unique, short pieces in a sequence specific
manner (not randomly)
• Named after the bacterium from which they are
isolated. [EcoRI- from E. Coli]
• Each enzyme recognizes & cleaves a specific &
double stranded DNA sequence
– 4-7 bp long
– Palindrome: same sequence from 5΄3΄ direction

8. Restriction site
• If recognize 4bp, cut once
in every 256 bp (4⁴)
• If recognize 4bp, cut once
in every 4096 bp (4⁶)
• The DNA cuts results in
– Blunt end
– Sticky end: useful in
constructing hybrid/
chimeric DNA molecule
• Restriction enzyme and
DNA ligase are used to
prepare chimeric DNA
molecules.

9. Library
A library- is a collection of recombinant clones
– Genomic library: prepared from total DNA of a cell
line or tissue
– cDNA library: only expressed DNA [cDNA copies of
mRNA] in a tissue

10. DNA cloning
• Production of a large number of identical copies of
DNA of interest by introducing that foreign DNA into a
replicating cell.
• Cloning vector
– DNA molecule to which the target DNA is joined
– Plasmid (present in all bacteria), Virus (phages)
– Artificial- cosmid, BAC, YAC
• Essential properties of vector
– Autonomously replicated within host cell
– Contain at least one specific restriction site
– Carry gene for selection (antibiotic resistance gene)

11. Steps of cloning
1. Restriction enzymes (e.g. EcoRI) cut the DNA to
be amplified
2. Bacterial plasmids (circular DNA additional to a
bacteria’s regular DNA) are cut with the same
restriction enzyme producing complementary
sticky end
3. DNA ligase joins the DNA fragment of plasmid
& DNA of interest to form a rDNA molecule
4. The recombinant plasmid is introduced into
host bacteria by transformation

12. 5. Bacteria are grown (in presence of antibiotic- so
only transformed bacteria survive, the others
die)
6. Bacteria are lysed, hybrid plasmid are isolated
7. Plasmids are cut with same restriction enzyme.
Many copies of DNA of interest released

14. Probes
• Probe is a molecule used to detect the presence of
a specific fragment of a DNA or RNA.
• Short piece (15-20 NT) of ssDNA or RNA, labeled
with a radioisotope [³²P]or fluorescent dye
• Complementary to a sequence of the DNA of
interest/ target DNA
• Used to screen a library for a complementary
sequence in the coding region of the gene.
• If the sequence match exactly, probes will hybridize

15. Probes

16. Blotting & hybridization
• Blotting is a process of immobilization of sample
nucleic acids on solid support (nitrocellulose
membrane)
• The blotted nucleic acids are then used as targets in
the hybridization experiments for specific detection
• Autoradiography allow visualization of specific
DNA/RNA fragments
• Types-
– Southern blot (for DNA)
– Northern blot (for RNA)
– Western blot (for protein)

17. Blotting & hybridization

18. 1. Southern blot: A method of transferring DNA
from an agarose gel to nitrocellulose filter, on
which the DNA can be detected by a suitable
probe [cDNA or RNA]
2. Northern blot: A method of transferring RNA
from an agarose gel to nitrocellulose filter, on
which the RNA can be detected by a suitable
probe [cDNA or RNA]
3. Western blot: A method of transferring protein
to a nitrocellulose filter, on which the protein
can be detected by a suitable probe [antobody]

19. Blot transfer procedure

20. Determination of DNA sequences

21. Polymerase chain reaction
• PCR is a enzymatic, test tube method of
amplifying a target sequence of DNA
• PCR can amplify
– Very small quantity of DNA (1 part in million)
– DNA from any source: bacterial, viral, plant
– Very rapidly (each cycle 5-10 min; 20 cycle made
10⁶ copies
– DNA from a single cell, hair follicle, spermatozoon

22. Steps of PCR
• Use DNA polymerase from Thermus Aquaticus [TaqP].
Heat stable, so not denatured at 70-80⁰C.
• Primer construction [20-35 NT] according to flanking
sequence
1. Denaturation of DNA [94⁰C]
2. Annealing of primers to ssDNA [54⁰C]
3. Chain extension by DNA-P [72⁰C]
New dsDNA molecules can be denatured & copied
repeatedly

23. Steps of PCR

24. Autoradiograph

25. Uses of PCR
• Forensic analysis of DNA sample
• To detect infectious agent [latent virus-HIV]
• To make prenatal genetic diagnosis
• To detect allelic polymorphism
• To establish precise tissue types for transplants
• To study evolution, using DNA from archeological
samples
• Quantitate RNA analysis [RT-PCR]
• To score in vivo protein DNA occupancy using
chromatin immuno-precipitation assay to facilitate NGS
[next generation occupancy]
New uses are developed every year.

26. Polymorphism
• Change in genotype that does not affect phenotype or
may change phenotype that is harmless.
• Primarily occur in non-coding sequence [only 2%
genome encode protein in human]
• Genome of any 2 unrelated people are 99.5% identical
Important tool in
– genome mapping
– localization of genes for genetic disorders
– determination the risk for disease
– paternity testing, criminal identification

27. Types of polymorphism
1. SNPs – single nucleotide polymorphism
 Single base changes in DNA
 Most common – 1 in 350 bp
2. RFLP- Restriction fragment length polymorphism
3. STR- Short tandem repeats (Microsatellite/ simple)
4. VNTRs- variable number tandem repeats

28. RFLP
When genome variation affect
restriction enzyme cleavage
site.
Detect human genetic variation
VNTR
VNTR is a location in a genome
where a short nucleotide
sequence is organized as
tandem repeat

29. Applications of rDNA technology
• Helps in gene mapping, thus localizes specific gene
on chromosome
• Understanding molecular basis of a number of
disease. e.g.- Familial hypercholesterolemia, sickle
cell disease, thalassaemia, cystic fibrosis, muscular
dystrophy and also some complex multifactorial
diseases (heart disease, cancer, DM)
• Commercial preparation of protein & hormone-
insulin, growth hormone, TPA, blood clotting
factor, interferon, interleukin

30. • Production of human enzyme used in therapy of
storage disease (e.g. Pompe’s, Gaucher’s disease)
• Preparation of several vaccines (Hepatitis B)
• DNA based diagnostic test can identify structural
variants (point mutation), dysfunctional & absent
genes (sickle cell disease, AIDS)
• This technology is used both to diagnose existing
disease as well as to predict the risk of developing a
specific disease and individual response to
pharmacological therapeutics
• Special techniques have lead remarkable advances in
forensic medicine

31. • Make platform for sequencing of genomic DNA
• Gene therapy cure a number of inherited disorder
& disease caused by somatic mutations (sickle cell
disease, thalassemia, Duchene muscular
dystrophy)
• Used in a number of analytic techniques- PCR,
RFLP, DNA typing, Southern b, northern b, western
blotting
• Trans genesis- of plant & animal to make more
resistant to disease & infection
• Huge application in agricultural & livestock
industries