| Studying Unculturable Microbes with PCR |
Imagine yourself on a team studying archaea at a deep-sea hydrothermal vent at the Galapagos Rift (an area known for its hydrothermal activity). You've found a new microbe. What do you want to know about it? What metabolic class does the microbe fall within? Does it make certain proteins? How does it survive the volcanic heat? Traditionally, asking such questions involved growing microbes in the laboratory. Unfortunately, replicating the conditions in which many bacteria and archaea grow is very difficult. For this reason, only a small fraction (perhaps only as few as one percent) of the microorganisms in nature has been cultivated. To identify and compare unculturable organisms microbiologists have turned to molecular genetic techniques.
Polymerase chain reaction (PCR) is one technique for studying organisms that cannot be grown in the laboratory. When only a small quantity of DNA is available from a particular source, PCR can be used to amplify that DNA and produce billions of copies of a designated gene-sized fragment. The technique has many applications, including the amplification of DNA from crime scenes, analysis of cancer genes, and identification of pathogens. When an environmental sample contains unculturable organisms, scientists can use PCR to generate copies of microbial genes suitable for comparison.
To replicate DNA in vitro, PCR takes advantage of a special property of the molecule: the hydrogen bonds. These bonds, which bind the complementary strands of DNA together in a double helix, are broken at elevated temperatures (about 95° C). Each single-stranded piece of DNA (ssDNA) is then built upon to form a new, double-stranded molecule (dsDNA). To initiate this, short "primers" -- specific ssDNA fragments called oligonucleotides -- must anneal to complementary regions on the single-stranded DNA. Deoxynucleotides (A,T,G, and C) and DNA polymerase are added and, in a process called primer extension, the complementary copy of the ssDNA fragment is built. The result is two double-stranded DNA molecules identical to the original. Repeating these steps thirty times can result in a 109-fold amplification of the original molecule.
Careful thermal cycling is required for PCR to proceed. For the primers to anneal to the ssDNA fragments, the temperature is reduced to about 55° C. However, at this temperature the original complementary ssDNA fragments will begin to re-anneal with each other. A high concentration of primers, and the tendency of the shorter primer strands to anneal more readily, ensures primer binding. The temperature is then raised again to about 72° C for primer extension. Underscoring the importance of microbes, the thermophilic bacteria Thermus aquaticus is the major source of the heat-tolerant DNA polymerase, which catalyzes primer extension and facilitates PCR.
In order to amplify a particular gene, specific primers, unique to that gene, are used. Two oligonucleotide primers (oligos) are constructed to flank a region of interest. One oligo will be complementary to a region on one strand of DNA, and the other oligo will be complementary to a region downstream on the homologous strand.
Back home, after your trip to the deep-sea hydrothermal vent, you want to determine what genus of bacteria you have in hand. You can use PCR to amplify the gene for ribosomal RNA (the gene isolated and sequenced by Woese from so many organisms when he constructed his "Tree of Life"). Then, you can choose conserved regions of the rRNA gene for primers. With adequate DNA from PCR, you could sequence the gene and compare it with millions of known rRNA gene sequences using a computer database. (See the Genomics unit.)
Alternately, you might want to ask if a microbe carries out a particular form of metabolism. Given the DNA sequence for a protein involved in a particular metabolic strategy - photosynthesis, for example - you could construct oligos so that the presence of that gene could be detected using PCR.
How does your microbe withstand the high temperatures of its volcanic environment? This has been a question posed by researchers studying extreme thermophiles for some time. Indeed, organisms have been found that tolerate temperatures as high as 110° C. Some archaea produce unusually high concentrations of thermoprotective proteins (heat shock proteins), which are found in all cells. These proteins help refold partially denatured proteins. Other archaea produce unique proteins that help stabilize DNA. You could use PCR to detect the genes for such proteins in your samples.
As the techniques of molecular genetics are applied to extreme environments we will come closer to understanding the wide variety of strategies that organisms use to survive on this planet... and perhaps on others.