Recombinant DNA technology allows DNA to be artificially combined from different sources. It involves isolating the target DNA and vector, cutting them with restriction enzymes, joining them together with DNA ligase, transforming them into host cells, cloning the recombinant DNA by cell replication, and selecting cells containing the inserted gene. Recombinant DNA has applications in producing pharmaceuticals like insulin and vaccines, genetically modifying organisms, gene therapy, and more.

1. Recombinant DNA Technology
Prepared By:
Shankar Thapa
M. Pharm (Pharmaceutical Chemistry) - 1St sem.
Department of Pharmaceutical Chemistry
Nargund College Of Pharmacy

2. Content:
➢ History
➢ Introduction
➢ Preview
➢ Procedure
➢ Identification
➢ Analysis
➢ Applications

3. History Of rDNA Technology
1. Watson and Crick (1953) : Discovery of DNA structure.
2. Isolation of DNA ligase in 1967.
3. Isolation of RE in 1970.
4. Paul Berg generated rDNA technology in 1972.
5. Cohen & Boyer (1973) : produced first plasmid vector capable of being replicated
within a bacterial host.

4. Introduction
▪ Recombinant DNA(rDNA) is a form of artificial DNA that is created by combining
two or more sequences that would not normally occur together through the process
splicing.
▪ Recombinant DNA technology , which is also called gene cloning or molecular
cloning , is a technique which allows DNA to be prepared via artificial means through
number of experimental protocols.

5. Preview
1. Gene to be cloned.egInsulin
2. Amplification – Insertion of the target gene in a host ( E.coli).
3. Extract DNA protein.
source: www.pubmed.com

6. Procedure
Steps of rDNA technology:
1. Isolating ( Target gene and Vector)
2. Cutting (Cleavage)
3. Joining ( Ligation)
4. Transformation
5. Cloning ( Amplifying)
6. Selection

7. 1. Isolating DNA
 For target DNA
Cells → disrupting lipid membranes with detergents → destroying proteins with
phenol or protease → degrading RNAs with RNAase → leaving DNA at the end.
 For vector
Bacterial cell → alkaline lysis or boiling → remove bacterial chromosomal DNA
from plasmid DNA → bacterial plasmid

8. Vector ( Plasmid)
 Vectors are vehicles and they contain sequence that allow them to be replicated
autonomously within a compatible host cell.
 All cloning vectors have unique cloning site, a sequence that can be cut by a RE
to allow site specific insertion of foreign DNA.
 They should have selectable marker.
 Types of vectors
1. Plasmid vector eg. pBR322
2. Bacteriophage vector
3. Expression vector
4. Yeast artificial chromosome (YAC)
5. Bacterial artificial chromosome (BA)

9. Cont…
 pBR322 plasmid vector
I. Characteristic
 Origin of replication
 Unique restriction site
 Selectablemarker ( tetracyclineand ampicillin)
source: www.bio.maima.edu

10. Restriction enzyme (RE)
 It is DNA cutting enzyme. It cuts DNA at specific sequencee.g. EcoR1 cuts at GAATTC and
BamH1 cuts at GGATCC.
 They can be obtained from bacteriaand named after the organism which they were derived
➢ EcoR1 from Escherichiacoli
➢ BamHI from Bacillus amyloliquefaciens
➢ Sau3Afrom Staphylococcusaureas.
source:www.bio.Miami.edu

11. 2. Cutting DNA ( cleavage)
 DNA can be cut into large fragments by mechanical shearing.
 It is done by Restriction endonuclease ( Restriction Enzymes)- RE
 RE will recognize specific nucleotide sequence in DNA and cut at Palindromic
site.
 These cut can be sticky or blunt end.
5’..GAATTC…3’
3’…CTTAAG…5’ palindromic
E.g. of ER
 EcoRI
 Bam HI
 Sau3A

12. 3. Joining DNA ( Ligation)
 The insert DNA and Vector are mixed in a tube and join by enzyme called DNA
ligase.
 Since both the DNAs have been cut with the same RE , the end will match up
because they are sticky.
 DNA ligase creates a phosphodiester bond between two DNA ends.
 After this , we transfer the rDNA into compatible host cells and the process is
known as transforming.

13. 4. Transformation
• rDNA transfer to the host ( bacteria) strain in a medium → treated with Cacl2
→ heated at 42 degree Celsius → rDNA goes into cell by a somewhat unknown
mechanism.

14. 5. Cloning ( Amplifying the rDNA)
• once in a cell , the rDNA will be replicated. When the cell divides the replicated
rDNA molecules go to both daughter cells which themselves will divide later.
• Thus , DNA is amplified.

15. 6. Selection
 It is the process of identification of host cells that contain rDNA of interest.
 Vectors usually contain some genetic marker, or gene, that allow the selection of
host cells that have taken of foreign DNA (rDNA).
 This selection can be done by introducing some antibiotic ( tetracycline,
ampicillin ) into the medium because some of the vectors are resistant to bacteria.

16. Identification of the specific gene of interest in the library
 1. Probing for the gene
a. DNA probe
DNA probe are based on the fact that a denatured DNA molecule will hybridize(bind)
to sequence that match or similar to it.

17. Cont…
b. Protein probe
protein product of the gene of interest → make an antibody against it → use the
antibody to protein of interest to screen the library .
2. Complementation
clone can be detected based on their ability to confer a missing function on a
mutant.
3. Positional cloning
is a method of cloning that makes use of information about a gene’s chromosomal
location in order to clone it.

18. Complementation
 Strain with mutation in Leu gene + DNA library
transformation
One of the cells in this culture will have received the plasmid with the Leu gene.
Plate on minimal media to select the clone containing
The Leu gene
Leu
gene
leu

19. Analysis of cloned genes ( screening)
1. Gel electrophoresis :
DNA fragments of different sizes can be separated by an electrical field
applied to gel. The negatively charged DNA migrates away from the negative
electrode and to the positive electrode. The smaller the fragment the faster it migrate.
It allows separation of vector DNA from cloned fragment.
2. Restriction enzyme mapping:

20. Cont…
3. PCR ( polymerase chain reaction)
▪ This technique is used for the sequencing the gene.
▪ Allow the isolation of a specific segment of DNA from small DNA (or cell sample)
using DNA primers at the end of the segment of interest.

21. Cont…
4. Blotting technique
• Blotting technique permit detection of specific DNA fragments and mRNA with DNA
probes.
a. Southern blotting
• First nucleic acid blotting procedure developed by Southern in 1975.
• Identified the DNA molecule.
b. Northern blotting
• It is the techniques for specific identification of RNA molecules.
• RNA on a gel with a DNA probe molecule.

23. Application of rDNA technology
 Analysis of gene structureand expression . E.g. GENE BANK.
 Pharmaceuticalsproducts
a. Drugs: eg. Insulin, interleukins, interferons, GH, erythropoietin, ,coagulation factor VII.
b. Vaccine: eg. Hepatitis vaccine
 Geneticallymodified organisms ( GMO)
i. Transgenic plants: Golden rice, insects resistant tomato
ii. Transgenic animal :
 Applicationsin medicine
 Human gene therapy.
 Diagnosis of genetic disorders.
 Forensic evidence.
 Application on environment like production of plastic degradation enzymes.

25. Reference
 A text book of pharmaceuticalbiotechnology by Springer publication.
 Molecularbiotechnology-principleand applicationsof rDNA by Bernard R. Glick and J.
Pasternak.
 www.bio.Miami.edu
 www.pubmed.com