1. Identification and Isolation of Desired Gene: The desired gene or DNA segment is identified and for its isolation restriction endonuclease enzyme is used.
Restriction endonuclease enzyme was discovered by Werner Arber and Hamilton O. Smith (1970). Restriction Endonuclease:
1. These enzymes are just like molecular scissors, which cut DNA at specific sites.
2. These enzymes are found naturally in bacteria such as E.coli, Bacillus, Streptococcus and Thermus aquatics.
3. Restriction endonuclease enzymes are of three types:
1. Type I Endonuclease (RI)
2. Type II Endonuclease (R-II)
3. Type III Endonuclease (RIII). Type II.
Note: Restriction endonuclease (RII) is most commonly used in gene cloning and restriction mapping. Example: E.co.RI, Hind II etc.
Nomenclature of Restriction Endonuclease:
1. The first alphabet of the name of the enzyme represents the genus from which it has been isolated. This is always written in capital letter.
2. The two alphabets after this represent the species of that genus. These are written in small letters. Note: All the three letters are written in italics Example: Eco from Escherichia coli. Hin – From Haemophilus influenza.
3. The fourth word represents the strain of the genus from which the enzyme has been isolated. Example: Eco R – From R strain of E.coli.
4. In case more than one restriction enzymes are obtained from one organism, their number is indicated by Roman numbers. Example: Eco RI, (First from R strain of E.coli) Eco RII etc. (Second from R. strain of E.coli) Endonuclease enzyme Eco RI recognizes specific base sequence in DNA molecule and cuts between the base G and A.
Note: When DNA segments obtained from two different sources are mixed together in the presence of DNA ligase enzyme, then both DNA segments are joined together by phospho-di-ester bonds and form a double-stranded structure.
Other Enzymes used in Genetic Engineering:
1. RNA dependent DNA Polymerase – This enzyme brings about polymerisation of nucleotides of DNA strand on an RNA template.
2. DNA dependent DNA Polymerase – This enzyme brings about polymerisation of nucleotides of complementary DNA strand on a DNA template.
3. Ligase – This enzyme joins the ends of DNA segments on the template.
4. Lysozymes – These enzymes dissolve the cell wall of bacteria so that the bacterial DNA can be easily isolated.
5. Alkaline Phosphatases – This enzyme cuts the phosphate at the 5′ end of circular DNA and helps to keep it in a linear form so that the foreign DNA segment can be inserted in it. This enzyme prevents the circular nature of DNA.
2. Selection of Cloning Vectors:
The desired gene is isolated with the restriction endonuclease. This desired gene containing a segment of DNA is then incorporated in a suitable vector. This vector should be able to enter into the target host or receptor cell and should replicate it’s DNA in the host cell.
Essential features of Vector:
1. It can be inserted easily into the host cell and can be isolated easily.
2. It should freely replicate in the host cell.
3. The vector must possess specific Restriction sites so that restriction enzyme can cut the DNA at those sites and the desired DNA fragment can be incorporated easily.
4. It should have one marker gene or marker site which can help in the selection of recombinants cells.
5. Transformation by it should be easy and perfect.
6. For expression of desired foreign DNA, the vector should essentially possess components such as a promoter, operator regulating sites.
Note: In E.coli both natural, as well as man-made vectors, can be used.
Some important vectors used in E.coli are:
1. Plasmid
2. Bacteriophage
3. Cosmid
