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What are the steps involved in the construction of an rDNA molecule for the purpose of gene cloning ? 

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Recombinant DNA (rDNA) : It is a form of DNA that does not exist naturally, which is created by combining DNA sequences that would not normally occur together. In terms of genetic modification, recombinant DNA is introduced through the addition of relevant DNA into an existing organismal DNA, such as the plasmids of bacteria, to code for or alter different traits for a specific purpose, such as antibiotic resistance. Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule.

• Treat DNA from both sources with the same restriction endonuclease (BamHI in this case). 

• BamHI cuts the same site on both molecules 

• 5′ GGATCC 3′ 

• 3’CCTAGG5′ 

• The ends of the cut have an overhanging piece of single-stranded DNA. 

• These are called “sticky ends” because they are able to base pair with any DNA molecule containing the complementary sticky end. 

• In this case, both DNA preparations have complementary sticky ends and thus can pair with each other when mixed. 

• DNA ligase covalently links the two into a molecule of recombinant DNA.

To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc., Producing many identical copies of the same recombinant molecule is called cloning. Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR). Here, however, we shall examine how cloning is done in vivo. 

Cloning in vivo can be done in :

• unicellular microbes like E. coli 

• unicellular Eukaryotes like yeast and 

• Mammalian cells grown in tissue culture.

In every case, the recombinant DNA must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. A number of viruses (both bacterial and of mammalian cells) can serve as vectors. But here let us examine an example of cloning using E. coli as the host and a plasmid as the vector.

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