Recombinant DNA technology involves joining DNA fragments from different sources to create a new DNA fragment. This involves selecting the target DNA, a cloning vector to carry the inserted DNA, DNA ligases to join the pieces, restriction enzymes to cut the DNA, and a host cell to replicate the new DNA. Common vectors include bacterial plasmids and phages that can carry inserted DNA fragments of varying sizes. The process involves cutting, joining, and replicating the DNA within the host cell. Applications include gene therapy, transgenic plants and animals, and industrial production of proteins and chemicals. Recent advances allow modifying proteins through site-directed mutagenesis.

1. DR SOURYA MOHAPATRA
1ST YR PGT DEPT OF
PHARMACOLOGY,SCBMCH
RECOMBINANTDNA -
TECHNOLOGY

2. *INTRODUCTION
It is a molecular biotechnology technique in which DNA fragments
from different sources are joined together to create a newly
modified DNA fragment of desired interest .
 Also K/A recombinant DNA(rDNA) or a chimera.

3. Discovery

6. The hosts are the living systems or cells in which the carrier of
recombinant DNA molecule or vector can be propagated.

8.  Phages (bacterial viruses)
Viruses infecting bacterial
species and multiplying within
it.
 Larger fragments of DNA can be
cloned in cosmids,which combine the
best features of plasmids and phages.
 Bacterial plasmids are
small, circular, duplex
DNA molecules that exist
in bact.cells and Naturally
confer antibiotic
resistance to the host cell.
These vectors are often
derivatives of plasmid pBR322
and contain the
necessary transcription and
translation start signals.

9. Vector DNA Insert Size (kb)
Plasmid pBR322(most common) 0.01–10
Lambda charon 4A 10–20
Cosmid 35–50
BAC 50–250
YAC 500–3000
CONT…

11. The various general steps involved in the preparation of Recombinant DNA can be briefly
Summarized As:
(1) Selection of DNA of interest
(foreign or target or passenger DNA)
(2) A cloning vector (to carry inserted pieces of target DNA)
(3) DNA ligases
(which joins pieces of two different DNA molecules together)
(4) Restriction endonucleases enzymes mostly RE-TYPE2 is used.
(which makes internal cuts at specifics sites on DNA )
(5) Host (to replicate the vector containing foreign
DNA: Prokaryotic / Eukaryotic cell).
(6) Screening test for recombinants produced by insertion of
DNA.(PCR,DNA Sequencing,NAH,BLUE WHITE SCREENING).
STEPS IN rDNA FORMATION

14. CELL MEMEBRANE DEPENDENT PROCESS

19. PROTOPLAST FUSION

20. Of rDNA

21. USES:MEDICAL

22. r-DNA in GENE THERAPY

23. TYPES OF GENE THERAPY

24. CONT..

25. OTHERS
2. Development of Transgenic Plants:
EG: BT-cotton, resistant to bollworms is a glaring example.
3. Production of Transgenic AnimalS
Cow, sheep, goat – therapeutic; human proteins in their milk. Fish like
common carp, cat fish, salmon and gold fish contain human growth
hormone (hGH).
4. Industrial Applications:
In industries, recombinant DNA technique will help in the production of
chemical compounds of commercial importance, improvement of
existing fermentation processes and production of proteins from
wastes.

26. RECENT ADVANCES:
Second Generation Therapeutic Proteins (Muteins):
• By employing site-directed mutagenesis, the amino acid sequence
of a recombinant protein can be suitably modified as desired, by a
technique referred to as protein engineering. The mutated
proteins are collectively referred to as muteins.
• Protein engineering is a rational approach to modify a protein with
regard to its stability, solubility, specificity, substrate affinity,
pharmacokinetics etc.
• The muteins obtained by protein engineering technique are
considered as Second generation of therapeutic proteins.
• Selected examples of such proteins (e.g. insulin lispro, Alteplase).

27. REFERENCES
• 1. R.MURRAY,D.BENDER HARPER’S BIOCHEMISTRY,28e,p388-
404.
• 2.U.SATYANARAYANA,U.CHAKRAPANI BOOK OF
BIOCHEMISTRY,3rde.
• 3.GG 13e.

28. THANK YOU