The slides describe restriction endonucleases, Polymerase chain reaction, cDNA and genomic library, vectors used in rDNA technology.

1. RECOMBINANT DNA TECHNOLOGY
Mr. A.T. Sharma
Assistant Professor
Nanded Pharmacy College, Nanded

2. Introduction to Biotechnology
• Two terms – Biology and Technology
• Biotechnology means any technological
application that uses biological systems, living
organisms, cells, tissues, explants, or
derivatives thereof, to make or modify
products or processes for specific use.
• Karl Erkey – Father of Biotechnology

3. Pharmaceutical Biotechnology
• Combination of two branches – Pharmaceutical science
and Biotechnology
• Applications of both industrial and medical biotechnology
• Defined as the science that covers all technologies required
for production, manufacturing and registration of biological
drugs.
• Involves using micro-organisms, macroscopic organisms, or
hybrids of tumor cells and leukocytes, to create –
- New pharmaceuticals
- Safer and/ or more effective versions of conventionally
produced pharmaceuticals

4. Applications of Pharmaceutical Biotechnology

5. Recombinant DNA Technology
• Manipulation of the genetic material towards
a desired end in direct and pre-determined
way.
• rDNA Technology/ Gene cloning/ Genetic
Engineering

6. Basic Steps
• Insertion of a specific piece of ‘desired DNA’ into a host
cell in such a way that the inserted DNA is replicated
and transferred to daughter cells during cell division.
• Isolation of desired gene
• Cutting of DNA into fragments
• Selection of vector
• Insertion of desired DNA into vector
• Transfer of recombinant vector into a host cell
• Selection of transformed host cells
• Growth of transformed cells

8. Objective
• Large No. of copies of specific DNA fragment
• Large quantity of protein
• Expression of genes
Vectors: Autonomously replicable DNA molecule
Tools used for gene cloning
• Restriction endonuclease
• Suitable DNA molecule capable of self replication
(vector)
• DNA ligase

9. Restriction Endonucleases
• Produce internal cuts called cleavage in DNA
molecule – Endonucleases
• Cuts within or near the sites with specific base
sequence – Restriction Endonucleases
• Sites recognized by RE – Recognition sequence
or site
• Types: Type-I, Type – II, Type - III

10. Type – I RE:
• Complex, RS – 15 bp
• Cleavage about 1000 bp away from 5’ end of the sequence
‘TCA’ in RS
• E.g. EcoK, EcoB
Type – II RE:
• Stable and cleavage within or immediately outside
RS, Symmetrical
• E.g. Eco57I, NgoMIV
Type III RE:
• Intermediate between type-I and II enzymes
• Cleave in immediate vicinity of their RS
• E.g. Ecop1, EcoP15

11. Nomenclature of RE
• First letter – Name of genus – capital
• Followed by first two letters of species name
in italic
• Strain or type as subscript e.g. EcoK
• More than one enzyme – Roman numericals
e.g. HindII, e.g. HindIII
• Suffix - R e.g. RHindII

12. Recognition Sequences
• Sites where DNA is cut
• Type II re – 4,5,6 bp rich in GC
• Palindromes with rotational symmetry
(ROTOR, MADAM)
Cleavage pattern
• Staggered cuts
• Blunt or flush cuts

13. Staggered Cuts
• Two strands of DNA cut at different locations
generating protruding (3’ or 5’) end.
• Rotational Palindromic RS – Protruding ends –
Complimentary base pairs – cohesive or sticky
ends
Blunt/ Flush Cuts
• Two strands of DNA cut at same locations –
Blunt ends

14. Staggered & Blunt Cuts

15. DNA Fragment Generation
• cDNA Library
• Genomic Library
Construction of cDNA Library
Definition: It is a population of bacterial transformants in which
each mRNA isolated from an organism or tissue is represented as
its cDNA insertion in a plasmid or phage vector.
Isolation of mRNA:
• Isolation of total RNA by extraction
 Bacterial lysis with trizol
 Bacterial lysis by hot SDS (Sodium dodecyl sulphate)
incubation
 Hot SDS hot phenol RNA method

16. • Concentrating the sample with desired mRNA
 Chromatography on poly-U Sepharose or oligo-T cellulose
 Density gradient centrifugation
 Using precipitated polysomes by specific antibodies
 Using specific cells with specific genes
e.g. Insulin gene in β-cells of pancreas
Preparation of cDNA
• mRNA – Template
• Reverse transcriptase (RNA dependent DNA Polymerase)
• Primer with 3’-OH group (Oligonucleotide)
• Eukaryotic mRNA as a template – poly-T oligonucleotide as a primer as
these mRNA have poly-A tail at 3’ end
• Prokaryotic mRNA - poly-A added to 3’ end by enzyme poly-A polymerase
• Annellation between primer and mRNA
• Base-pairing continued by reverse transcriptase
• RNA – DNA hybrid molecule production

17. • Digestion of RNA strand by RNase-H or
alkaline hydrolysis - single stranded DNA
• End of DNA acts its own primer
• A hair pin loop-like structure produced –
cleaved by single strand specific nuclease

19. Applications of cDNA Library
• Discovery of novel genes
• Cloning of full length cDNA molecules for in-vitro
studies
• Study of different kinds of mRNA expressed in
different cells or tissues
• Study of alternative splicing in different cells or
tissues
(A process where by multiple RNA transcripts
are generated from a single gene.)

20. Genomic Library
• A collection of plasmid clones or phage lysates
containing rDNA molecules so that the sum total of
DNA inserts in this collection, ideally, represents the
entire genome of concerned organism.
Construction
• Total genomic DNA of organism extracted by
 Cell lysis by chemical or physical methods like blending,
grinding, sonication
 Remove membrane lipids by surfactants
 Remove proteins by adding proteases
 Precipitating DNA with an alcohol

21. • DNA cut in to fragments by mechanical
shearing, sonication or RE
• Partial digestion by RE with 4 base RS – more
appropriate sized fragments
• Separation of appropriate sized fragments by
agarose gel electrophoresis or sucrose
gradient centrifugation
• Isolation of desired fragment done by using a
labelled probe
• Fragments inserted in to vector – cloned in to
host

22. Vectors
• DNA molecule that has ability to replicate autonomously in an
appropriate host cell and in to which the DNA fragment to be
cloned (DNA insert) is integrated for cloning. E.g. plasmid, phage,
virus etc.
Properties:
 Replicate autonomously
 Size – less than 10 kb
 Easy to isolate and purify
 Easy transformation of host
 Easy detection and selection of transformed hosts
 Ability to integrate in to host cell
 Cells transformed by rDNA should be identifiable from cells
transformed by vector only
 Unique target sites for as many RE as possible
 Suitable control genes

23. Types of Vectors
Cloning vectors:
• A small piece of DNA in to which a foreign
DNA can be inserted for cloning purpose
• Used for propagation of DNA inserts in a
suitable host
• Contains features that allow for the
convenient insertion or removal of a DNA
fragment to or from the vector
• E.g. plasmids, bacteriophages etc

24. Expression vectors:
• Plasmid or virus used for expression of genes, i.e.
protein synthesis
• May be fusion protein or a pure protein
• Contain regulatory sequence necessary for
controlling protein synthesis
E. coli Vectors
• E. coli as a host
• Easy cloning and isolation of DNA
• Initial expression carried out in E. coli
• E. coli strain K12 is most commonly used

25. Plasmids
• Extra-chromosomal, double stranded, circular, self-
replicating DNA molecules
• Size: 1-500 kb
• 0.5 – 5% of total DNA of a bacteria
Nomenclature:
• First letter – p for plasmid
• Followed by first letter of researcher’s name and then
numericals given by workers
• E.g. pBR 322 – Bolivar and Rodriguez – designated as 322
• According to place of discovery
• E.g. pUC – University of California

26. pBR322
• Most popular E. coli
vector, parent or grand
parent
• DNA sequence of 4361
bp
• Marker genes – genes
for ampicillin and
tetracycline resistance
• Unique RS for enzymes –
EcoRI, HindIII, BamHI,
SalI, PstI etc.

27. Requirements for a Plasmid Vector
• Less than 10 kb size
• Relaxed replication control
• At least two selectable markers
• RS for restriction endonuclease
• RS located within one or two selectable
markers

28. Bacteriophages

29. λ-phage vector
• Viruses that infect bacteria
• Lytic or lysogenic cycles
• Lysogenic (temperate phages)– integrate with bacterial
chromosome and multiply- prophage
• Lytic phages produce plaques – clear, bacteria-free zone
• DNA insert up to 53kb
• E.g. λ-phage vector
• Most studied and developed E. coli vector
• Head and tail – hypodermic syringe
• DNA – 50kb, linear with cohesive ends
• DNA cyclises to form circular DNA
• Insertion vector: DNA size 5-11kb
• Replacement vector: DNA size 8-24kb

30. Polymerase Chain Reaction (PCR)
• Laboratory technique – large quantities of DNA
• Cell-free amplification technique
• Karry Mullis in 1984 – Nobel prize in 1993
Principle
• Denaturation – Two strands separate
• Renaturation – Base pairing between primer and
template
• Synthesis – DNA synthesis
• Three steps repeated many times – multiple
copies of target DNA

31. Requirements
• A target DNA
• Two primers
• Four deoxyribonucleotides
• Taq DNA polymerase enzyme (thermostable)
Stages
• Denaturation: Temperature used: 95οC –DNA strand separation
• Renaturation/Annealing: Temperature used: 55οC
- Primer – template base pairing
- High primer concentration
• Synthesis: Initiates at 3’-hydroxyl end of primer
- Primer extension – temperature: 75οC
- Reaction stopped by increasing temperature: 95οC
• Formation of long and short templates
• 32 cycles – a million fold DNA

33. Applications
• Clinical diagnosis
• DNA sequencing
• Comparative study of genomes
• Forensic studies

34. Production of Interferons
• Earlier, blood as a source of interferons
• Use of bacterial hosts – relatively less
Steps:
 Isolation of mRNA
 Construction of cDNA
 Fragmentation of DNA
 Isolation of desired DNA
 Amplification of gene of interest
 Ligation
 Transfer of rDNA into host cell
 Screening
 Culturing of transformants
 Extraction
 Downstream processing (Recovery and purification)

36. Production of Hepatitis B Vaccine (Recombivax)
Steps:
• HBs antigen producing gene isolated
• Plasmid DNA extracted and cut
• Insertion of HBs producing gene in vector
• Introduction of rDNA in to a yeast cell
• Fermentation
• Extraction of yeast cells
• Purification

38. Production of rDNA Insulin

39. Steps:
• Hakura and Colleagues (1977) synthesized DNA
sequence of insulin for two chains A and B and
separately inserted in to two plasmid vectors
pBR322
• Genes inserted by side of β-galactosidase gene of
plasmid
• Recombinant plasmids separately transformed in
to host – E. coli
• Formation of pro-insulin chains separately
• Pro-insulin chains separated from β-galactosidase
• A and B chains joined by sulphonation

41. Thank You…!!!