Prepare a flowchart in the formation of recombinant dna by the action of restriction endonuclease,EcoRI

• Plasmid (autonomously replicating, circular, extra-chromosomal DNA) is isolated.

• Plasmid DNA acts as a vector since it is used to transfer the piece of DNA attached to it to the host.

• Plasmid DNA also contains genes responsible for providing antibiotic resistance to the bacteria.

• Plasmid DNA was cut with a specific restriction enzyme (‘molecular scissors’ − that cut a DNA at specific locations).

• The DNA of interest (to be inserted) was also cut with the same restriction enzyme.

• The DNA of interest is hybridized with the plasmid with the help of DNA ligase to form a recombinant DNA.

•  Recombinant DNA is then transferred to a host such as E.coli, where it replicates by using the host’s replicating machinery.

• When E.coli is cultured in a medium containing antibiotic, only cells containing recombinant DNA will be able to survive due to antibiotic resistance genes and one will be able to isolate the recombinants. 
  
  Action of restriction enzymes: 
  Every restriction enzyme identifies different sequences (Recognition sequences). Over 900 restriction enzymes have been isolated, all of which recognize different sequences.

• Recognition sequences are pallindromic-  Palindromes are the sequence of base pairs that read same both backward and forwards (i.e., same and direction). 
  Example: ­­

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• Restriction enzymes cut a little away from the center of palindrome site, but between the same two bases on the opposite strands.

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• As a result, overhangs (called sticky ends) are generated on each strand.

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• Sticky ends form hydrogen bonds with their complementary counterparts with help of DNA ligases.

• All these process es form the basis of RDT.

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Regards.