01-08-2012, 03:40 PM
RECOMBINANT DNA TECHNOLOGY AND ITS APPLICATION
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INTRODUCTION
Definition of recombinant DNA technology:-
A series of procedures used to recombine DNA segments under certain conditions, a recombinant DNA molecule can enter a cell and replicate.
Organism DNA+Foreign DNA=Production of recombinant DNA,such organism are known as Transgenic organism.
History of recombinant DNA technology
Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973.
Basic principle of recombinant DNA technology
The DNA is inserted into another DNA molecule called ‘vector’.
The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced.
Gene cloning
It can be defined as the isolation and amplification of an individual gene sequence by insertion of that individual gene sequence into a bacterium where it can be replicated.
There are some steps involved in gene cloning:-
1.Isolation of gene of interest
2.A fragment of DNA to be cloned is incorporated into a small replicating DNA molecule called a vector.
3.The recombinant vector is introduced into a host cell by transformation.
4.Cell that contains rDNA are selected.
5.Growth and multiplication of rDNA.
Enzyme for cutting and joining the dna
Cutting DNA molecule at the specific sites with restriction endonucleases.
There are three types of restriction endonuclease:-
TYPE I:-they are complex nucleases.
TYPE II:-they are simple enzymes that consist of a single polypeptide.
TYPEIII:-they contain two different subunit and require ATP and Mg++ as cofactor.
Joining of dna molecule
The enzyme used to join DNA fragments are called DNA ligase.
This is the final step in construction of a recombinant DNA molecule,process is called Ligation.
Bacteriophage vectors
Bacteriophage are viruses that infect bacteria.
These are usually called phages and it is constructed from two basic components: protein and nucleic acid genome.
Phages can be double stranded (T2,T4,T6,lambda) or single stranded(phage 174,M13).
There are both DNA(T2,T4,T6) and RNA(MS2) phages.
Phages contain an origin of replication.
The process is known as transduction by which phages enter into the bacteria.
Ti-plasmid
Most cloning vectors for plants are based on the Ti plasmid,which is not a natural plant plasmid but belongs to a soil bacterium Agrobacterium tumefaciences.
This bacterium invades plants tissue,causes a cancerous growth called a crown gall.
During infection,a part of Ti plasmid called the T-DNA is integrated to the plant chromosomal DNA.
There are Ri plasmid present in A.rhizogenes which causes hairy root disease in plants.