The construction of artificial recombinant DNA molecules is termed as genetic engineering or gene manipulation, sometimes also referred to as gene cloning or molecular cloning because it produces a line of genetically identical organisms, all of which contain the same recombinant DNA molecule.
The major steps involved in genetic engineering are:
- Production and isolation of the DNA fragments to be cloned.
- Insertion of the isolated gene in a suitable vector to obtain recombinant DNA.
- Introduction of the recombinant DNA into a suitable organism/cell (usually E.coli) called host (transformation).
- Selection of the transformed cells, and identification of the clone containing the desired gene/DNA fragment.
- Multiplication/expression of the introduced gene in the host.
- Where needed, transfer and expression of the gene into another organism.
The tools used for genetic engineering are those biological products or agents which are used to achieve its objectives. Four different types of enzymes are used viz. nucleases, ligases, polymerases, and certain DNA modifying enzymes.
Some of the other tools used are listed as below:
- Restriction endonucleases to the DNA at specific sites; often known as molecular scissors.
- A suitable Vector.
- DNA ligase to seal the nicks that remain in recombinant DNA molecule; often known as molecular glue.
- A suitable host for the propagation of the recombinant DNA molecule (DNA insert).
- Reverse transcriptase is used to produce cDNA (complementary DNA).
- Alkaline phosphatase for removing 5’-phophate from the DNA ends.
- T4 polynucleotide kinase for addition of phosphate group to have an ends having a free 5’-OH.
- SI nuclease for removal of single stranded protrusions from ends; both 3’- and 5’- extensions and removed.
- The Klenow fragment of E. Coli DNA polymerase I to make the protruding ends double-stranded by extending the shorter segment.
- Lambda exonuclease for removal of nucleotides from the 5’-ends.
- E.coli exonuclease III for removal of nucleotides from 3’-ends.
- Exonuclease Bal31 for making DNA fragments blunt ends shorter from both its ends.
- Terminal deoxynucleotidyl transferase for addition of single stranded sequences to 3’- ends of blunt-ended frangments.
- Linker and adapter oligonucleotide sequences for modification of the cut ends of DNA fragments.
Four different types of enzymes are used viz. nucleases, ligases, polymerases, and certain DNA modifying enzymes.
- For the production and isolation of the DNA fragment one of the following methods may be used:
- For the insertion of the gene into a suitable vector either of these methods are used:
- For further introduction of the recombinant DNA molecule into the host cell one of these methods is suitable:
- The end process of screening for the desired clones is carried out in one of the following manner:
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