cDNA — Cut, Paste, and Copy Genes -
We talked about the role of cDNA in the BRCA gene story and how they were used by scientists to discover the gene and how to assemble its sequence (you can find that post here if you missed it). Now we will talk more about the main advantages of working with cDNA.

cDNAs are small pieces of DNA that are easily moved around. Whole libraries of genes can be transported and used in many important ways with cDNAs, as in the pictures at the top right. cDNAs express genes and can be moved into and out of cells and animals. The middle and bottom pictures show a protein that glows green when illuminated with UV light placed in cells and rats using cDNAs.
But how is that done?
Living things make special enzymes called endonucleases that are like molecular scissors. They cut large pieces of DNA into little pieces, but in a very specific way. The scissors only cut when the DNA has exactly the correct sequence of a small number of nucleotides (A, C, T, G) called a recognition sequence. There are many types of these scissors, almost a thousand, each with its own recognition sequence. This allows a scientist to cut out just the the small piece of DNA that they want and isolate it easily in a test tube. The discovery of endonucleases was one of the most important discoveries in the early days of DNA research, and earned Werner Arber, Daniel Nathans, and Hamilton O. Smith the Nobel Prize in 1978.

Endonucleases are enzymes that act like molecular scissors cutting strands of DNA at specific places.
Modified from Understanding DNA and Gene Cloning, Drlica 1984
So what do you do with all these short DNA fragments?
In short, we can glue them together in a useful order using another enzyme, called a ligase, that acts like molecular tape pasting the pieces together (like fitting together the BRCA gene that we talked about earlier).

After DNA is cut with endonucleases, the fragments can be pasted together using another enzyme, ligase.
This patchwork cDNA is then inserted into a circular DNA structure called a plasmid that lives in bacteria using endonucleases (the scissors) and ligases (the tape). Plasmids hang out in bacteria and whenever the bacterial cell divides the plasmids are copied. Because bacteria can grow millions and millions of cells very quickly, lots and lots of plasmids each with a copy of the cDNA can be generated in a short amount of time.

The patchwork cDNA is then pasted into a ring of DNA called a plasmid.

This plasmid is then transferred into bacteria which makes a copy of our cDNA every time they divide.
The bacteria are like microscopic plasmid factories, all making the plasmid with our cDNA of interest. Finally, it is easy to isolate the plasmid from the bacteria and Huzzah! we have lots of cDNA.


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Originally published at lcresearchcenter.tumblr.com.
