Recombination DNA Technology

1. Recombinant DNA Technology
Presented
by
Merajun Alam
Roll:568
Batch: 19th
Semester: 5th
Department of Pharmacy

2. Contents
 Introduction
 Discovery
 Goals and objectives
 rDNA technology procedure
 Enzymes
 Vectors
 Techniques
 Applications

3. Recombinant DNA technology
DNA molecules that are extracted from different sources and
chemically joined together is called recombinant DNA and this
technology is called Recombinant DNA Technology . Forexample
DNA comprising ananimal gene may berecombined with DNA
from a bacterium

4. Discovery of recombinant DNA
technology
Discovery of DNA structure Watson & Crick in 1953
Isolation of DNA ligase in 1967
Isolation of REase in 1970
Paul Berg generated rDNA technology in 1972
Cohen & Boyer in 1973 produced first plasmid vector
capable of being replicated within a bacterial host

5. Goals of recombinant DNA technology
• To isolate and characterize a gene
• To make desired alterations in one or more isolated genes
• To return altered genes to living cells
• Artificially synthesize new gene
• Alternating the genome of an organism
• Understanding the hereditary diseases and their cure
• Improving human genome

6. Procedure of making rDNA
Isolating of DNA
Cutting of DNA
Joining of DNA
Amplifying of DNA

7. Isolating of DNA

8. Cutting of DNA
• DNAcan be cut into large fragments by mechanical
shearing.
• Restriction enzymes are the scissors of molecular
genetics.

9. Restriction enzyme
• Aspecial class of sequence-specific enzyme
• Found in bacteria
• Site-specific-cleave DNAmolecules only at
specific nucleotide sequence
• REases recognize DNAbase sequencethat
are palindrome
• REase make staggered cuts with
complementary base sequences for easy
circulization

10. Joining DNA

11. Amplifying the recombinant DNA
• Transforming the recombinant DNAinto a bacterial host strain.
• The cells are treated with CaCl2
• DNAis added
• Cells are heat shocked at 42 C
• DNAgoes into cell by a somewhat unknown mechanism.
• Once in a cell, the recombinant DNAwill be replicated.
• When the cell divides, the replicated recombinant molecules go to both
daughter cells which themselves will divide later. Thus, the DNAis
amplified

12. Amplifying the recombinant DNA

13. Enzymes used inrecombinant DNAtechnology
• Bind to DNAmoleculesDNA ligase
• Cleaves DNA at specific sitesType II restriction endonuclease
• Make a DNA copy of RNA moleculeReverse transcriptase
• Fill single stranded gapes of DNAduplexDNA polymerase I
• Adds a phosephate to the 5'-OH end of a polynucleotidePolynycleotide Kinase
• Adds homopolymer tails to the 3'-OH endsTerminal transferase
• Removes nucleotide residues from the 3' endsExonuclease III
• removes nucleotides from the 5' endsBacteriophage {lamda} exonuclease
• Removes terminal phosphatesAlkaline phosphatase

14. Vectors used in rDNA technology
vectors
BACS
YACS
express
ion
cosmid
Lamda
phage
plasmid

15. Techniques used in rDNA technology
• Gel electrophoresis
• Cloning libraries
• Restriction enzyme mapping
• PCR
• Nucleic Acid Hybridization
• DNAMicroarrays

16. Applications of rDNA technology
• Pharmacology: artificial insulin production, drug delivery to
target sites
• Medicine: gene therapy, antiviral therapy, vaccination,
synthesizing clotting factors

17. Agriculture
Growing crops of your choice
(GM food), pesticide resistant
crops, fruits with attractive
colours, all being grown in
artificial conditions

18. Other uses:
Fluorescent fishes,
glowing plants etc