Gene Cloning Process in rDNA Technology
i. Isolation of DNA (gene) from the donor organism:
a) Extract and purify DNA from the donor organism.
ii. Cutting of the desired gene:
a) Use restriction enzymes (restriction endonucleases) to cleave isolated DNA.
b) Cleaved DNA fragments have cohesive or blunt ends.
c) Select a fragment containing the desired gene, now termed foreign DNA or passenger DNA.
iii. Insertion of a desired foreign gene into a cloning vector (vehicle DNA):
a) Integrate foreign DNA into a cloning vector, often plasmids or bacteriophage viruses.
b) Plasmids, such as pBR322, are isolated from bacterium vector organisms.
c) Cleave vector DNA with the same restriction enzyme used for gene isolation.
d) Use DNA ligase to insert foreign DNA, forming Recombinant DNA or Chimeric DNA.
iv. Transfer of rDNA into suitable competent host or cloning organism:
a) Transfer recombinant DNA into a competent host cell, typically a bacterium.
b) Host cell incorporates rDNA into its chromosomal DNA through ‘transformation’.
c) Divalent Ca++ assists rDNA transfer.
d) Cloning organisms in plant biotechnology include E. coli and Agrobacterium tumefaciens.
e) Transformed cell is termed a transformed cell.
v. Selection of the transformed host cell:
a) Use marker genes on the plasmid vector for differentiating transformed from non-transformed host cells.
b) Example: pBR322 plasmid with Ampicillin resistant and Tetracycline resistant genes.
c) PstI restriction enzyme knocks out Ampicillin resistant gene, making recombinant cells sensitive to Ampicillin.
vi. Multiplication of transformed host cell:
a) Separate and cultivate transformed host cells.
b) Introduce cells into fresh culture media, allowing multiplication and replication of recombinant DNA.
vii. Expression of the gene to obtain the desired product:
a) Induce transformed host cells to express the gene, leading to desired product production.
b) Separate and purify the product through downstream processing in a suitable bioreactor.
Outline of the process of recombinant DNA technology