The Recombinant DNA Molecule Into The Host Cell
a) Transfection with recombinant phage DNA.
The ? (alpha) phage vectors can be used as naked DNA and introduced into E.coli cells just as is plasmid DNA by heat shock following CaCl2 treatment the method known as transfection. The phenomenon of transfection is identical to transformation, except that in this case phage DNA is introduced into the bacterial cell in place of plasmid DNA.
b) Transformation with recombinant plasmid DNA.
Extrachromosomal replicons which are stablely inherited existing as double-stranded circular molecules are known as plasmids. The procedure through which a DNA is introduced in a host is known as transformation, it includes the process like micro-injection and biolistic.
c) In vitro packaging into phage coat: transduction with recombinant phage or cosmid.
Plasmids which include the cos site are termed as cosmids and they are used as gene cloning vectors in conjugation with the in vitro packaging system. The recombinant cosmid DNA is injected and circularizes like phage DNA but replicates as a normal plasmid without the expression of any phage functions. The transformed cells are selected on the basis of a vector drug resistance marker.
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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