Clone DNA

Cloning DNA is a technique to produce large amounts of DNA. Often this is done using bacteria. To clone a specific DNA sequence, one must first place the DNA into a bacterium. This is done by either inserting the DNA sequence into a plasmid or virus and forming a recombinant DNA molecule. To insert the DNA of one organism into another requires the use of restriction enzymes. Once inside the bacterium, the DNA is replicated by the bacterium (plasmid) or the virus.

How do you know that the recombinant plasmid was taken up by the bacterium? Well, you could add a second gene that confers antibiotic resistance. Once the bacteria have been stimulated to take up the recombinant plasmid, you could grow the bacteria on a plate containing an antibiotic. Those bacteria that survive have the new plasmid. There are several other ways to evaluate bacterial colonies with out disrupting the culture. The point is that once a culture containing the recombinant DNA is identified, these cells can be used to grow new cultures in quantity.

Once you have the quantity of cells you need, you then extract the DNA from the bacterium. Then you need to separate the bacterial DNA from the smaller plasmid DNA.

• What technique can you use to separate the plasmid DNA from the bacterial DNA?

Finally, you can use the same restriction enzyme you used to make the recombinant plasmid to remove the replicates or clones of the DNA sequence you desire from the rest of the plasmid DNA.

• What technique can you use to separate the plasmid DNA from the cloned DNA?

Another way to clone DNA is to use a virus.

• What advantages are there to using viruses to clone DNA instead of using bacteria?

Another way to clone DNA is to use messenger RNA.

• How can you use messenger RNA to clone DNA?

These methods of cloning DNA do not work well with DNA sequences larger than 20 to 25,000 base pairs.

• How would you clone large DNA sequences, say 100,000 to 1,000,000 base pairs?
• What type of restriction enzyme would you use: one that recognized four nucleotide sequences or one that recognized eight or more nucleotide sequences? Why?

DNA clones are used to make DNA libraries of an organism's genome.