This ppt shows the basics of rDNA technology and its earlier researches.

2. Recombinant DNA technology
 Altering the genome of an organism by introducing the
genes of interest is known as Gene manipulation or rDNA
technology .
 This mechanism has the ability to engineer new organisms
it is called Genetic engineering .

3.  It is the technique where the selected DNA of donor is
introduced to combine with the DNA of recipient organism.
As a result the recipient acquires the genetic abilities of the
donor.
History :
The rDNA technology is one of the
recent advances in biotechnology , which was developed by two
scientists Herbert.W.Boyer and Stanley.N.Cohen in 1973.

4. The DNA sequence used in the construction of
recombinant DNA molecule can originate from any species.
using this technology any DNA sequence may be created and
introduced into any of the living organisms.
ex : Human DNA may be joined to
E.coli.
ENZYMES :
Bacterial cells have different
kinds of enzymes.

5. RESTRICTION ENDONUCLEASES : (1970)
These are the scissors of
molecular genetics. Hence they are called as Molecular
scissors. Restriction enzymes are endonucleases that
recognize specific nucleotide sequence in the DNA and cut at
those points. Varieties of RE are commercially available . Most
cut at specific palindromic sites in the DNA . The cuts
produce “sticky or overhanging ends”.

6. DNA ligase :
 DNA ligase is the glue of Molecular genetics that holds
the ends of the DNA s together.
 It creates a phosphodiester bond between the two
DNA ends. Ligase is used in the final step of the
construction of the DNA molecule and the process is
called as Ligation.

8. Vectors : formation of rDNA requires cloning vector .
 It is generally derived from plasmids or viruses.
 It contain small segment of DNA that contain necessary
signal for replication.
Characters :
1. Capable of replicating in host cell .
2. Small and easy to isolate.
3. Have selectable marker for indication.
4. convenient RE sites for inserting the DNA of interest.

9. Plasmids :
 They are double stranded closed circular DNA molecules
which exist in a cell as a extra chromosomal unit.
 They are self replicating and replicate independently.
 size less than 20 kb if larger than 20 kb are lost easily
in bacterial cell.
ex : pBR322 , pUC19 .

11. Marker gene :
 It is used to know whether the DNA has been
successfully transferred into recipient cell.
 This is introduced into the plasmid along with the target
gene.
Two types :
1. Reporter gene .
2. Selectable markers.

12. Steps :
DNA of donor or gene of interest is isolated
Cut using Restriction endonucleases.
The donor DNA fragments are attached to vector.
The DNA of vector is cut into fragments using same RE .

13. the fragments are joined using DNA ligase.
Splicing.
As a result Hybrid DNA or rDNA is obtained.
 rDNA is introduced into host cells such as E.coli ,
Bacillus subtilis , Streptomyces sp.,

15.  The host cells are treated with cellulase so that the cell
wall become permeable to entry of rDNA .
 Host cells follows the instruction of rDNA .
 After a short time a colonies of bacteria having rDNA
fragments is produced.
 Each colony is grown seperately to produce identical
copies of rDNA fragments.
This is called molecular cloning .
Ex : Human insulin produced by E.Coli.

16. It has both advantages and disadvantages .
Advantages :
 used in Medical , agriculture , industry , food production ,
bio-engineering .
 Human growth Hormone (somatotropin)
 Human Insulin
 Golden rice (beta carotene)
 Herbicide resistant crops (Maize, sorghum etc.,) resistant
to herbicide Roundup.
 Insect – resistant crops .(beta toxin)

17. Disadvantages :
 Difficult to clone five or more genes which control a trait.
 Plant characters such as growth rate , size of edible parts,
amino acid balance are polygenic controlled by many genes .
Such traits are not yet identified.

18. Transformation :
The uptake of exogenous
DNA by cells that alters the phenotype or genetic trait of cell
is called as Transformation.
Griffith experiment :
In 1928 the Bacteriologist
Fredrick griffith Conducted experiments using Diplococcus
pneumoniae.

19.  S – Smooth – Virulent – polysaccharide capsule present –
Harmful .
 R - Rough - Avirulent – No capsule – Harmless .

21. Result :
 The heredity material of the heat killed S- type cells
transformed R-type cells into virulent smooth strains.
 The transforming factor was found to be DNA .
The amount of cell transformed per 1
micrograms of DNA is called Transformation efficiency.