RECOMBINANT DNA TECHNOLOGY AND APPLICATIONS

1. RECOMBINANT DNA TECHNOLOGY
CELLULAR & MOLECULAR PHARMACOLOGY
PRESENTED BY
JITHIN MATHEW
MPHARM FIRST YEAR
DEPT OF PHARMACOLOGY

2. RECOMBINANT DNA TECHNOLOGY
It is the technique of Joining together of DNA Molecules of two Different
species that are inserted into a host organism to produce New Genetic
Combination.
 Proteins That can results from the expression of Recombinant DNA Within
living cells are termed as Recombinant proteins
 Construction of Recombinant DNA in which a foreign DNA fragment is
inserted into a Plasmid vector

4. PRINCIPLESOF RECOMBINANT DNA TECHNOLOGY
 Gene Cloning & Development of Recombinant DNA
 Transfer of vector into the Host
 Selection of Transformed cells
 Transcription & Translation of inserted Gene

5. Gene Cloning & Development of Recombinant DNA
Gene to be cloned must be inserted in a cloning vector( Plasmid)
DNA Fragment is introduced into a bacterial cell & Replicated
Copying DNA @ specific site called origin of replication

7. Transfer of vector into host
Process of introducing purified DNA into Bacterial cell called
Transformation
Cell Treated with Cacl2 and high temperature
Transformed cells are obtained

8.  Extra chromosomal DNA That lack origin of replication
 uptake pf non plasmid DNA have no significance
 Suitable strains of E.coli is used

10. Selection of transformedcells
 Host cells are identified and take up the R DNA
 Cells are grown in medium containing ampicillin, tetracycline
shows resistance due to distortion of gene
 selected recombinant bacteria are grown in bioreactor to
obtain the gene product

12. Transcription and Translation of Insertedgene
Transcription of DNA into MRNA is mediated through RNA
polymerase enzyme
Recognise Binding site on DNA called promoter

13. Process of MRNA synthesis Terminated by a terminal signal(
Terminal codon) gene lying between promoter& Terminator will
transcribed
Isolated gene must be inserted into vector close to promoter
site

15. VECTORS
Human genes are introduced into bacteria at first gene is transferred into a
carrier known as vectors
TYPESOF VECTORS
1)PLASMIDS
2)CLONING VECTORS
A)Type1 bacteriophage
B) Type2 vectors
3) COSMIDS

16. PLASMIDS
 Plasmids are circular double stranded DNA molecules seen inside the
bacteria
Plasmid confer antibiotic resistance to host bacteria easily exchanged
between living bacteria
Antibiotic resistance property is exchanged in bacteria 8-20 copies of
plasmids seen inside bacterium
Plasmid replicate independent of bacterial DNA

18. CLONING VECTORS
1)Type1 Bacteriophage
 Kind of cloning vector carry up to 10 lakh of inserted gene
 Helpful to maintain large piece of DNA
 E.coli has the term lambda has been developed as a cloning vector

20. TYPE2 PHAGEMIDE VECTORS
A phagemid is a hybrid of a plasmid and a filamentous coliphage that can be
propagated in either form
 Coliphage should be either of the 3 virtually identical stages
These are male specific phage contains single stranded circular DNA as there
genome
 Infection of E.coli by the bacteriophage double stranded DNA is first formed
is replicate immediately

21. COSMID VECTORS
 Plasmid vectors are not suitable for cloning DNA fragment
very much longer than own size as the transformation
frequency fall beyond the acceptable limit
 They are often used as a cloning vector in genetic
engineering
They can also be packaged in phage capsids
 Foreign genes are transferred by transduction

22. RESTRICTIONENDONUCLEASE
 Selectively split the parent DNA by this enzyme
Also known as molecular scissors
Certain enzymes of bacteria restricts the entry of phage's into bacteria
called Restriction endonuclease
Named after the species & strains of bacteria and the order of
discovery

23. RESTRICTION SITE
 The enzyme recognise specific site where hey cut DNA
 There are more than 600 enzymes are available commercially.
 The enzyme recognise specific arrangement called Palindrome arrangement.
 Also called Inverted repeat sequence in 5‫׳3-׳‬ direction
 Resultant DNA have overlapping or sticky ends

24. ENZYME SOURCE OF ENZYME SPECIFIC SEQUENCE
IDENTIFIED BY
ENZYME
ECOR1 Eserenchia coli RY13 G
Hind111 Haemophilus
influvenza Red
AATT
TTAA
C
G
A AGCT T
T TCGA A

25. SUMMARY OF RECOMBINANT DNA TECHNOLOGY
Human m RNA
C DNA COPY
Plasmid vector cleaved by specific restriction
endonuclease

26. Transfection into host bacteria
Clone amplification
Isolation of protein

27. PHARMACEUTICALAPPLICATION
 Production of lymphokines
 Production of growth hormone
 Transgenic Application
 Production of vaccine
 Cloning of human artificial receptor for drug development& Design
 Production of insulin

28.  Diagnostic aids using RDNA
a)Hybridisation of Nucleic acid
b) Southern Blotting
 Detection of Genetic disorder

29. REFERENCE
 Textbook of Pharmaceutical Biotechnology
S.P Vyas & V.K Dixit Page no 341- 348
 Textbook of Biochemistry By D.M
Vasudevan & Srikumari Page no- 375- 377
 Harpers Review of Biochemistry

30. THANK YOU