(73)
4. Definere begrebet 'recombinant DNA'
Devlin, s.287-8, fig. 7.6
 

Recombinant DNA – combination of two DNA fragments of different origin into one DNA molecule by the use of restriction enzymes and DNA ligase.  

Recombinant DNAs have been prepared with combination of DNA fragments between bacteria, viruses, humans etc. The ability to join two different peaces of DNA together at specific sites within the molecules is achieved with two enzymes, a restriction endonuclease and a DNA ligase.
 

Restriction endonuclease are enzymes that recognize specific inverted repeat sequences in double-helical DNA and cleave, at specific places, both strands of a duplex  containing the recognized sequences. A specific restriction enzyme cuts DNA at exactly the same sequence site regardless if it is a bacterial, viral, mammalian or human DNA.

Many restriction endonucleases hydrolyze DNA in a staggered fashion, yielding fragments with single stranded regions on their 5’- and 3’-ends. DNA fragments generated from different molecules with the same restriction endonuclease have complementary single-stranded ends, which can be annealed and covalently linked together with a DNA ligase.

DNA ligase catalyses the formation of a phosphodiester bond between the 3`-OH group at the cleaved end of one DNA-species molecule and the 5´-phosphate group at the end of the other cleaved DNA from a different species. This of course happens on both strands, so we get a double helical recombinant DNA.

The DNA fragments produced from restriction endonuclease that forms blunt ends can also be ligated but with much lower efficiency.

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