The DNA which contains DNA from two different sources is called Recombinant DNA and the technology for the formation of recombinant DNA is called DNA technology.
Following materials are required for producing recombinant DNA:
- Gene of interest: The genes are to be cloned.
- Molecular scissors: These are used to cut out the gene of interest.
- Molecular carrier or vector: The genes of interest can be placed on it for transrport.
- Expression system: The gene of interest with the vector is introduced into an expression system. Thus a specific product is made.
(a Gene of interest
Th re are three possible ways to get the gene of interest.
- Isolation of gene from the chromosome: Genes can be isolated from the chromosomes by cutting the chromosomes on the flanking sites of the gene.
- Special enzymes restriction endonucleases are used to cut the genes.
- Chemical synthesis &genes: Small genes can be synthesized in the laboratory.
- Making gene from mRNA: It is a very common method of getting the gene.In this case, gene is synthesized in the laboratory from messenger RNA by reverse transcriptase enzyme.Molecular Scissors: Restriction Endonucleases
T e restriction enzymes are present in bacteria. The bacteria use these e ymes for their own protection against viruses. The restriction enzyme cuts d n the viral DNA. But they do not harm the bacterial chromosome. They re trict the grOwth of viruses. So they are called restriction enzymes.
H milton 0. Smith isolated the first restriction enzyme in 1970. These enzymes ct the DNA at very specific sites. These sites have specific sequence of four or
EcoRI is a commonly used restriction enzyme. It cuts double-stranded DNA at the specific sequence of bases. So a gap is produced in this
D A. A piece of foreign DNA w h complementary ends can be placed in this gape. The single stranded DNA with complementary ends of the o DNA molecules is called ” sticky ends” Thus they can bi d by complementary base painng. Therefore, the
restriction enzymes help in the insertion of foreign DNA in o vector DNA.
( ) Molecular Carrier Vector
T e means by which recombinant DNA is introduced into a host cell is c lied vector. A vector is selected to make recombinant DNA.
1 Plasmids: Plasmids are natural extra chromosomal circular DNA molecules. Plasmid is common type of vector. The investigators discovered Plasmids during their study of the sex .life of the intestinal bacterium, Escherichia coil. They carry genes for antibiotic resistance and fertility etc.
2. DNA of bacterial viruses: The DNA of bacterial virus can also be used as a vector. For example lambda phage. Lambda phage attaches to a host bacterium. The recombinant DNA is released from the virus and enters the bacterium. The recombinant DNA replicates and many copies of the viruses are formed. Each virus in bacteriophage clone contains a copy of the gene of choice.
(d) Expression System
Bacteria are mostly used as expression system. The clone of bacteria is prepared. A clone can be a large number of molecules (i.e. cloned genes) or cell (i.e. cloned bacteria) or organisms that are identical to an original specimen.
Process of synthesis of recombinant DNA
- The bacterial cells are treated with calcium chloride. It makes the bacterial membrane more permeable. Now the bacterial, cells take up recombinant
- These bacteria reproduce and bacterial clones are formed. Each new cell contains at least one plasmid. Therefore, each clone of bacteria contains thegene of interest,3 The clone bacteria express themselves and make a product.
4 The protein product can be separated from the clone bacteria.
5 The cloned gene can be isolated from this bacterial clone for further analysis.