Recombinant DNA Technology
and its Applications.
Basic Steps of Genetic Engineering
Step 1: DNA Extraction
Step 2 : Gene Cloning
Step 3 : Gene Design
Step 4 : Transformation
Step 5 : Backcross Breeding
Recombinant DNA (or rDNA) is made by combining DNA
from two or more sources.
What is Gene Cloning?
Gene cloning is a technique, where an isolated single
copy of a gene is cloned to obtain its indefinite identical
There are two types of gene cloning:
In vivo, which involves the use of restriction enzymes
and ligases using vectors and cloning the fragments into
The other type is in vitro which is using the polymerase
chain reaction (PCR) method to create copies of
fragments of DNA.
A. Steps in molecular cloning
1. Formation of recombinant DNA molecule includes:
(A) Selecting the host organism and cloning vector,
(B) preparation of DNA to be cloned,
(C) preparation of vector DNA and
(D) creation of recombinant DNA.
2. Transfer of recombinant DNA into host organism.
3. Selection and screening for positive clones.
Fig: Different steps involved in procedure to clone a gene
Creating Recombinant DNA
This involves joining of the broken ends of the vector DNA with the two ends
of DNA sequences to be cloned.
There are 3 methods of joining DNA fragments :
DNA ligase- joins the cohesive ends
T4 Ligase- forms phosphodiester bonds between blunt ended fragments.
Terminal deoxynucleotidyl transferase- synthesizes homopolymeric tails at the
ends of fragments.
B. Confirmation of Cloned Gene
1. By Gel Electrophoresis
2. Southern Blot - Specific genes can be identified
with DNA or RNA probes using the Southern Blot
Gene Cloning in Plants by Agrobacterium mediated
Fig: Agrobacterium transfers T-DNA into plant cell and induces tumour