Gene Cloning Technique

Gene Cloning Technique in Molecular Biology Field Involves the following steps:

1.a) Isolation of Genetic Material and Gene Sequence of Interest. 

First, cells are lysed using detergent or lysozyme enzymes which disrupts the plasma membrane and release the genetic material along with the macro molecules such proteins and RNA molecules. Cell contents are then treated with protease to disrupt the proteins and RNase to destroy the RNA. Cell debris are then pelleted using centrifuge and supernatant containing DNA is transferred to a fresh and clean tube. A proper amount of Ethanol is added to this supernatant and precipitated using centrifuge. Supernatant is discarded and the pellet which has DNA is suspended using a proper suitable buffer. 

Primers are designed for the specific gene sequence of interest which will be used for the cloning procedure. 

Gene sequence is then amplified using PCR (Polymerase Chain Reaction) which will yield in many copies of the gene sequence. 

1.b) Restriction Digestion and Ligation of Insert into a Vector. 

The amplified gene sequence is then treated with specific Restriction Enzymes to cut the gene sequence at specific sites and to make the sequence to have the sticky ends.

Same restriction enzymes are used to cut the plasmid (vector) at specific sites and to have the sticky ends. 

After treated the gene sequence and plasmid with same restriction enzymes, these two needs to ligated. 

In a suitable buffer, the restriction digested plasmid and gene sequence are added along with the Ligase enzyme. Ligase enzyme helps in joining the plasmid and gene sequence as they have the sticky ends. 

And then, it becomes the recombinant DNA molecule (Plasmid+Gene sequence). 

[if the plasmid is not already isolated...... the host cell which has the plasmid is treated with lysozymes and detergents (bufferes) to break open the cell, and the cell debris are treated with proteases and RNase to destroy the proteins and RNA. and cell debris are pelleted and supernatant containing the DNA is treated ethanol and purified. The pellet is then diluted using an appropriate buffer.]

2) Transformation of recombinant DNA into the host cell. 

After obtaining the recombinant DNA, this DNA needs to transferred or introduced into the host cell, where it can replicate or multiply into many copies of its own. Usually, a bacterium is used as a host cell to introduce this rDNA (although, other types of living cells can be used depending on the applications and questions). 

Commonly, used method is Transformation, in which the rDNA molecule is introduced into the host cells and these host cells are then grown on an agar plate at 37℃. (There are other methods also to introduce the bacterium carrying the rDNA molecule). 

3) Multiplication of rDNA molecule.

Within the host cells, the vector multiplies which in turn produces numerous identical copies, not only of itself but also the gene sequence that it carries. (Need to consider the proper temperature and other physical conditions required for the host cell to grow and multiply). 

4) Division of Host Cell.

When the host cell divides, copies of the recombinant DNA molecule are passed to the daughter cells or progeny and further vector replication takes place. 

5) Cell Divisions resulting in a clone.

After a large number of cell divisions, a colony of cells or a clone, of the identical host cells is produced. Each cell in the colony or clone contains the copies of the recombinant DNA molecule (Vector + Gene Sequence). 

The gene sequence of interest carried by the recombinant DNA is now said to be cloned. 

Image Source: gene cloning - Rent.interpretomics.co