Using the DNA Sequence
Once DNA is in a manageable form, its sequence can be read, studied, and even changed. Knowing the sequence of an organism's DNA allows researchers to study specific genes, to compare them with the genes of other organisms, and to try to discover the functions of different genes and gene combinations. The following are some techniques scientists use to read and change the sequence of DNA molecules.
Reading the Sequence Researchers use a clever chemical trick to “read” DNA by determining the order of its bases. A single strand of DNA whose sequence of bases is not known is placed in a test tube. DNA polymerase, the enzyme that copies DNA, and the four nucleotide bases, A, T, G, and C, are added to the test tube. As the enzyme goes to work, it uses the unknown strand as a template to make one new DNA strand after another. The tricky part is that researchers also add a small number of bases that have a chemical dye attached.
Each time a dye-labeled base is added to a new DNA strand, the synthesis of that strand is terminated. When DNA synthesis is completed, the new DNA strands are different lengths, depending on how far synthesis had progressed when the dye-tagged base was added. Since each base is labeled with a different color, the result is a series of dye-tagged DNA fragments of different lengths. These fragments are then separated according to length, often by gel electrophoresis, as shown in the figure at right. The order of colored bands on the gel tells the exact sequence of bases in the DNA.
Cutting and Pasting DNA sequences can be changed in a number of ways. Short sequences can be assembled using laboratory machines known as DNA synthesizers. “Synthetic” sequences can then be joined to “natural” ones using enzymes that splice DNA together. The same enzymes make it possible to take a gene from one organism and attach it to the DNA of another organism. Such DNA molecules are sometimes called recombinant DNA because they are produced by combining DNA from different sources.
Making Copies In order to study genes, biologists often need to make many copies of a particular gene. Like a photocopy machine stuck on “print,” a technique known as polymerase chain reaction (PCR) allows biologists to do exactly that. The figure below shows how PCR works.
Polymerase Chain Reaction Polymerase chain reaction (PCR) is used to make multiple copies of genes.
The idea behind PCR is surprisingly simple. At one end of a piece of DNA a biologist wants to copy, he or she adds a short piece of DNA that is complementary to a portion of the sequence. At the other end, the biologist adds another short piece of complementary DNA. These short pieces are known as “primers” because they provide a place for the DNA polymerase to start working.
The DNA is heated to separate its two strands, then cooled to allow the primers to bind to single-stranded DNA. DNA polymerase starts making copies of the region between the primers. Because the copies themselves can serve as templates to make still more copies, just a few dozen cycles of replication can produce millions of copies of the DNA between those primers.
Where did Kary Mullis, the American inventor of PCR, find a DNA polymerase enzyme that could stand repeated cycles of heating and cooling? Mullis found it in bacteria living in the hot springs of Yellowstone National Park—a perfect example of the importance of biodiversity to biotechnology.