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RECOMBINANT DNA TECHNOLOGY

Integration of isolated or artificially synthesized DNA into a vector to produce recombinant DNA is known as recombinant DNA technology. The recombinant DNA thus produced can be screened by the selection markers (e.g. antiobiotic resistance in the vector may be lost by integration of foreign DNA) (Fig.12.12) and separated by gelelectrophoresis from other DNAs (e.g. reannealed circular vector and foreign DNAs). This recombinant DNA is then transferred into a host (target) cell to generate useful products (e.g. insulin) or to produce transgenic organisms (e.g. disease resistant crop plants) to be used for the welfare of human beings. Whenever required, the foreign DNA can be retrieved by treating with the same restriction enzyme. Some of the important techniques for the generation of recombinant DNA, depending upon the requirement, are:

Fig-12.12

Fig.12.12. Screening of ampicillin sensitive recombinant clones by replica plating in ampicillin-containing medium.

Using Restriction Enzymes that Produce Staggered Cuts: The vector and the foreign DNAs are cut with a restriction enzyme separately to produce sticky complementary ends. When the two DNAs are mixed together, the complementary ends of the foreign DNA may anneal with the complementary ends of the vector DNA, which are joined finally by adding the enzyme, DNA ligase (Fig.12.13).

Fig-12.13

Fig-12.13

Fig. 12.13 Recombinant DNA technology using EcoRI, REI = Restriction Enzyme I.

2.Blunt End Ligation by T4 DNA Ligase: In this method, the plasmid DNA is made linear by cutting with a restriction enzyme to produce blunt ends. When artificially synthesized restriction site (linker DNA) of a restriction enzyme is added separately into the linear vector DNA and foreign DNA in the presence of T4 DNA ligase (which can link two double-stranded DNA segments with blunt ends), the linker DNA gets attached at the ends or vector DNA as well as to that of the foreign DNA. Now, the two DNAs are treated with the restriction enzyme that cuts the restriction site (linker DNA) to generate sticky complementary ends. After mixing the two DNAs, recombinant DNA is produced after ligation by the DNA ligase enzyme. This technique is also used ill III preparation of cDNA library (Fig. 12.14).

Fig-12.14

3. Adding Poly dG at the 3’ End of Vector and Poly dC at the 3’ End of Foreign DNA: The vector DNA after cutting with a restriction enzyme that generates blunt ends, is added with dGTP in the presence or enzyme terminal transferase to produce poly dG at the 3’

Fig-12.15

ends of this linear vector DNA (addition of new nucleotides during polymerization always occurs at the 3′ end) . Similarly, the foreign DNA is mixed with dCTP to generate poly dC at its 3′ ends. When the two DNAs are mixed together recombinant DNA is produced due to complementary dG : dC base pairing. This dG : dC is the restriction site of the restriction enzyme Pst I (obtained from Providentia stuartii and the foreign DNA, whenever required, can be retrieved by treatment with the same particular restriction enzyme (Fig. 12.15).