BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
Tools of Recombinant DNA technology
1. Restriction Enzymes - used to cut DNA at specific locations
2. Gel electrophoresis - used for the separation and isolation of desired DNA fragment
from a mixture of DNA fragments
3. Vector (Plasmid) - used for the transfer of desired DNA into a host
4. DNA ligase - enzyme used to join two DNA ( plasmid DNA with desired DNA
fragment) commonly called molecular glue
5. Host - cell / organism into which the recombinant DNA is transferred
Restriction Enzymes
• Commonly called molecular scissors.
• Restriction enzymes are found to be restricting the growth of bacteriophages by cutting its DNA.
• Restriction enzymes cut the DNA at specific locations
• Restriction enzymes are isolated from different strains of bacteria
• The first isolated restriction enzyme was Hind II.
Naming of restriction enzyme (Eg : EcoRI )
First letter (E ) – represent the genus name of bacteria ( Escherichia)
Next two letters (co) - represent the species name of bacteria (coli)
Forth letter (R) - represent the strain of bacteria (RY 13)
Roman numeral (I) - represent the order of isolation (first)
• Restriction enzymes belongs to the category Nuclease
• Restriction enzymes are two types namely exonuclease and endonuclease. Exonuclease cut the DNA
from ends (ie, remove nucleotides from ends) while endonuclease cut at specific locations within
the DNA. Example for endonuclease is EcoRI and Hind II.
• Restriction endonuclease cut at specific locations called recognition sites and the particular base
sequence at the recognition site is called recognition sequence.
• The recognition sequence of EcoRI is GAATTC.
• Recognition sequence is palindromic sequence also. Palindromic sequence is the sequence of base
pairs that reads same on the two strands when orientation of reading is kept same. So the
palindromic sequence of EcoRI is 5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
• After finding the recognition sequence, restriction enzyme binds to the DNA and cut the strands a
little away from the centre of the recognition sequence between the same two bases on the
opposite strands. This creates overhanging single stranded portions at the ends and is called sticky
ends. When cut by the same restriction enzyme, the DNA fragments and plasmid have same kind of
sticky ends and can be joined together using DNA ligase.
GEL ELECTROPHORESIS
• Gel electrophoresis is used for the separation and isolation of desired DNA fragment form a mixture
of DNA fragments.
• DNA fragments are negatively charged molecules and are separated by forcing them to move
towards the anode through the agarose gel under an electric field.
• The commonly used gel is agarose and is obtained from sea weeds
• Agarose gel act as the medium / matrix for the movement of DNA fragments
• The DNA fragments move according to their size. Smaller fragments move farther ie, move more
towards the anode side
• The separated DNA fragments can be visualized by staining with Ethidium bromide followed by
exposure to UV light. Now the DNA fragments appear as bright orange coloured bands
• The desired DNA fragments are cut out from the agarose gel and extracted from the gel piece. This
step is called Elution
Cloning Vectors
The vectors can carry a foreign DNA (desired DNA) and replicate inside the host cell. Vectors may be
Plasmids, Bacteriophages, retroviruses etc. the best known of these vectors is the plasmid vectors. Plasmids
BR322
are the extra chromosomal DNA of some bacteria. The p is the first artificial cloning vector developed
from E. coli plasmid. A plasmid has the following characters when it is used as a vector in rDNA technology.
a. Origin of Replication (ori)
This is a sequence from where replication starts and any piece of DNA when linked to this
sequence can replicate within the host cells. It is also responsible for controlling the copy
number of the linked DNA.
b. Selectable Marker
It helps to identifying and eliminating the non-transformants and permitting the growth of
R R
transformants. Antibiotic reststant genes such as amp and tet are used as selectable
BR322
marker in p .
R
amp - Amphicilin resistant gene
R
tet - tetracycline resistant gene
c. Cloning Sites (Recognition sites/target sites)
To link the foreign DNA, the vector requires recognition sites for the restriction enzymes.
Usually a vector has a single cloning site for a particular restriction enzyme.
Competent Host
DNA being a hydrophilic molecule cannot pass through cell membranes. The bacteria should be
made competent first, to accept the DNA molecules by treating them with a specific concentration of a
divalent cation like calcium which increases the efficiency with which DNA enters the cell through the pores
in its cell wall. Recombinant DNA can be forced into such cells by incubating the cells on ice, followed by
placing them briefly at 42OC and then putting them back on ice. This facilitates s the entry of recombinant
DNA in the bacterial cell. This method is called heat shock method. Other methods are micro injection, gene
gun and disarmed pathogen vectors. In microinjection method rDNA is directly injected into the nucleus of
an animal cell. In gene gun or Biolistic method cells are bombarded with high velocity micro-particles of
gold/tungsten coated with DNA in plants. Disarmed pathogen vectors like Ti plasmid and retroviruses are
also used to transfer desired DNA into host
Processes of recombinant DNA technology
Process of rDNA technology involves the following steps:
Isolation of DNA
↓
Fragmentation of DNA by restriction enzyme
↓
Isolation of desired DNA fragment by gel electrophoresis
↓
Ligation of the desired DNA fragment into a vector
↓
Transferring the rDNA into a host
↓
Culturing the host cells in a medium at large scale
↓
Extraction of the desired product
