Genetic Engineering, Part I

It seems like barely a day goes by without a news story having to do with manipulating genes, moving genes from one organism to another or the impact of these kinds of genetic manipulations. The key points of genetic engineering are quite simple and stem from the description of the DNA double helix that was proposed by Watson and Crick. Advances in genetic engineering have much to do with learning how to apply what was learned long ago.

So, genetic engineering consists basically in cutting and pasting DNA. How can you cut and paste a molecule of DNA? The technical term for this is to "make recombinant DNA". “Recombinant DNA” refers to the combination of DNA from two different sources. Our ability to create recombinant DNA in the lab is based on a fortuitous discovery having to do with how bacteria defend themselves from being attacked by viruses. Many kinds of viruses specialize in attacking bacterial cells, and they are called bacteriophages. They attach to the outside of the cell and inject their DNA into it. Then, the virus’ genetic material takes control of the cellular machinery of the bacteria, turning it into a factory for making more copies of the virus.

This is very bad news for the bacteria, so you can imagine that natural selection would favor the evolution of mechanisms that defend the bacteria against viral attack. One such mechanism, discovered in the late 1960’s, involves enzymes produced by the bacteria, called “restriction endonucleases”, or simply restriction enzymes. They cleave the double helix of the DNA molecule, breaking it into two pieces. In so doing, they render the DNA non-functional. The viral DNA that’s injected into the bacterial cell is chopped up before it can take control of the bacteria. This is an effective defense, but there is in fact a problem here. If restriction enzymes produced by the bacteria can cut up a molecule of DNA, what’s to stop these enzymes from attacking the bacteria’s own DNA?

Part of the answer to this question is exactly what made restriction enzymes so useful to molecular biologists. Restriction enzymes don’t cut the DNA double helix in random places, but only at a precise point defined by a particular sequence of bases. These sites are called “recognition sites”, or “restriction sites”.

Restriction Sites

Restriction sites are typically only a few nucleotides long, about four or six bases long. Another interesting characteristic of these sites is that the two complimentary strands of DNA usually are palindromic. In language, a palindrome refers to a set of letters that are spelled the same way backwards or forwards. For example, the word “dad” is a palindrome, as is the sentence “Madam I’m Adam”. In DNA, a palindrome occurs when the two strands of the double helix have the same sequence of bases in reverse direction with respect to each other.

So, if the sequence of one strand is GAATTC, then the complimentary sequence on the opposite strand would be CTTAAG. It is indeed like this because of the complimentary rules that Watson and Crick discovered so long ago.

How does the fact that these restriction enzymes only cut DNA at specific palindromic sequences help prevent the enzymes from chopping the bacteria’s own DNA? This actually doesn’t help the bacteria directly, because given that these sequences are so short, it’s very likely that somewhere in the bacteria’s own DNA that sequence would occur. However, what the bacteria can do is to selectively protect the restriction sites found in its own genome by slightly modifying the bases.

Specifically, what happens is that so-called methyl groups are added to some of the bases occurring at the restriction sites of the bacteria’s own DNA. This “methylation” prevents the enzyme from identifying that area as a restriction site, and thus protects the bacteria’s own DNA.

So, how does this help us make recombinant DNA? The answer to this question relies on one more fact we have to learn about restriction enzymes. Not only does the restriction enzyme recognize particular sequences of nucleotides, it also cuts the DNA strands very precisely between just two particular nucleotides in the sequence. Because the restriction site is palindromic, the exact place where the cut is made on each of the complimentary strands of the double helix would be offset from each other by a few bases.

If the restriction site sequence of one strand of DNA is GAATTC, then we know that the sequence on the opposite complimentary strand would be CTTAAG. A restriction enzyme recognizing this site might cut the DNA exclusively between the G and A nucleotides. In this case, after the double strand is cut, each cut piece would now have a short section of single stranded DNA.

The exposed bases on these single stranded bases of the cut DNA molecule are called sticky ends. They are called sticky ends because they would line up, form complimentary base pairs, and essentially stick to any other single stranded sequence of bases having the complimentary sequence. Here’s the key point, the sticky ends from any fragment of DNA that had been cut using the same restriction enzyme would always be complimentary to each other by definition. This means that if you cut two different DNA double helixes with the same restriction enzyme, even DNA from completely different species, when you mix all of those fragments of DNA, they would come back together because of the base pairing.

Pasting the DNA

Then, you add to the mixture the enzyme DNA ligase. This enzyme sticks together the newly joined DNA helixes by building back the strong chemical bonds that the restriction enzyme had broken.

Once you’ve used DNA ligase to put the sticky ends back together permanently, you now have a piece of recombinant DNA that is made of two different original molecules.

There are hundreds of different kinds of restriction enzymes that had been isolated from a variety of bacteria. Each different kind recognizes a different DNA sequence. So, they cleave DNA at a different point along the overall length of the molecule. By using different restriction enzymes, you can cut molecules of DNA in different places, and then paste them back together in different arrangements. Actually in practice there are many other details that you have to consider to make this process work. In theory, at least, our ability to cut and paste DNA boils down to the use of enzymes normally used by bacteria to defend themselves against viruses.