TOOLS OF RECOMBINANT DNA TECHNOLOGY

BIOTECHNOLOGY: Principles and processes

DEFINITION:
Recombinant DNA Technology (RDT) is joining together DNA molecules from two different
species that are inserted into a host organism to produce new genetic combinations that are of
value in industries, medicine, agriculture, and research.

Recombinant DNA Technology has made it possible to isolate a single gene and enabling
researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly
specific ways, and reinsert the modified sequence into the living organism.

TOOLS OF RECOMBINANT DNA TECHNOLOGY (RDT):

1. Restriction enzymes
2. Cloning vectors
3. A competent host

RESTRICTION ENZYME
DEFINITION: Special type of enzymes that cuts DNA molecule from specific nitrogenous
base. Examples or restriction enyzmes are:
- Hind II - Hind III
- Eco RI - Etc.

TYPES OF RESTRICTION ENZYMES:

1. Restriction endonuclease
2. Restriction exonuclease

RESTRICTION ENDONUCLEASE
- Type of restriction enzyme which cuts DNA at specific nucleotide
sequences at interior side

RESTRICTION EXONUCLEASE
- Cuts DNA at specific nucleotide sequences but at the exterior side of
DNA. It removes nucleotides from the ends of DNA.
 You can see in this picture that it is the vector DNA
and foreign DNA. With the help of restriction
enzyme EcoRI, we will get the sticky end after cutting
both vector DNA and foreign DNA. And, with the help
of enzyme DNA ligase, the DNA fragments join at
sticky ends, and finally, you will get the recombinant
DNA.

CLONING VECTORS
DEFINITION: DNA molecule which can freely do replication in appropriate host and in which
desired DNA segments can be inserted. Examples are:
- Plasmid - Bacteriophage
- Cosmid - Etc.

CHARACTERS OF A GOOD VECTOR:

1. Should bear origin of replication (ori) site due to which it is able to multiply within
the host cell. It represents the sequence from where replication initiates.
2. Should contain a selectable marker (usually antibiotic resistance) that should be
absent in host main chromosome
3. Should not have the tendency of natural recombination so that recombinant
DNA remains unchanged.
4. Methylase should be absent. This makes the recognition site of R.E. resistant by
methylation. Due to this, there is more problem in manipulation of recombinant
DNA.

These are all the main characters of a good vector. But no natural DNA molecule is a
good vector. Thus, vectors used in cloning are produced by attaching useful segments
of different natural DNA molecules. By this, some most useful and new vectors are
obtained.

You can see, this is the picture of pBR322

vector. It is the most famous artificial
vector. It is 4362 bp (base pair) long and
its whole sequence is known. Having
restriction sites for Hind III, EcoR I, BamH
I, Sal I, Pvu II, Pst I, and also Cla I.
COMPETENT HOST:
- Host like bacteria, yeast, plant, and animal cell can be used, and methods to
introduce DNA into host cell may be Gene gun method, Microinjection, or
Electro-poration.