🎨 Recombinant DNA Technology


Making DNA
Cloning DNA
Applications


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Making DNA

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Say we are interested in the gene for insulin. Sure, we could take it straight from people, but remember humans are eukaryotes and eukaryotes have non-coding sequences within their genes called introns. The mRNA following splicing, on the other hand, has no introns! How can we make DNA from mRNA?

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Take this arbitrary bit of mRNA: UCCAUGCCAUUUGGG

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If we had an enzyme which could reverse the transcription back into DNA, this time intron-free, that would be great. We do – it’s called reverse transcriptase and it produces DNA. This special case of DNA is called complementary DNA – cDNA.


cDNA via reverse transcriptase: AGGTACGGTAAACCC (remember that DNA unlike mRNA is double-stranded; not shown for simplicity)Β 

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If we wanted the portion after the second G above, is there a way we could cut the DNA? It appears so. Some microorganisms have actually evolved enzymes whose job it is to invade a host and chop its DNA up at specific sequences. These enzymes are called restriction endonucleases. Each has its own short sequence which it recognises. There is a restriction endonuclease called CviQI which has the recognition site GTAC and cuts between G and T. That fits our bill!

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The other DNA strand will also have a GTAC site read in the opposite direction. Notice that the complementary sequence of GTAC backwards (starting from the C) is…