Submitted by

T.Tamilselvan

Construction of rDNA molecules

and Bacterial transformation

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 .

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.

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.

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”.

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.

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.

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 .

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.

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 .

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.,

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.

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)

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.

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.

S – Smooth – Virulent – polysaccharide capsule present – Harmful .

R - Rough - Avirulent – No capsule – Harmless .

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.

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