PCR equipment. Courtesy of the Microbial Ecology/Biogeochemistry Research Laboratory, NASA Ames Research Center.
Not all microorganisms can be grown in culture. In fact, microbiologists have figured out how to grow very few species in the lab, less than 1% of all the microbial species in the world.
PCR allows scientists to extract and analyze bits of microbial DNA from samples, meaning they don’t need to find and grow whole cells.
PCR is an essential element in DNA fingerprinting and in the sequencing of genes and entire genomes.
A schematic of how the PCR process works. Courtesy of Guruatma "Ji" Khalsa, M.N.S.
Basically, it’s like a technique to photocopy pieces of DNA. In a matter of a few hours, a single DNA sequence can be amplified to millions of copies.
PCR lets scientists work with samples containing even very small starting amounts of DNA. The technique makes use of the DNA repair enzyme polymerase. This enzyme, present in all living things, fixes breaks or mismatched nucleotides in the double-stranded DNA helix. These breaks or mismatches could cause genes to malfunction if left unfixed.
Polymerase uses the intact half of the DNA molecule as a template and attaches the right nucleotides, which circulate constantly in the cell, to the complementary nucleotide at the site of the break. (DNA consists of two strands of nucleotide bases, which are represented as A, G, C, and T. In the laws of DNA base-pairing, A joins with T and G with C.)
Not all polymerases are created equal, however. Many fall apart in high heat. PCR was developed in 1985 following the discovery of an unusual heat-loving bacterium called Thermus aquaticus in a hot spring in Yellowstone National Park. This bacterium’s polymerase, dubbed Taq, does its job of matching and attaching nucleotides even in the high heat generated by the successive “photocopying” cycles required during PCR. Taq made PCR possible.
16S rRNA. Courtesy of the Helix Group at the Stanford Section on Medical Informatics.
Scientists also use molecular tools to extract and compare bits of a particular kind of RNA from samples in order to determine if previously known or new microbes are present in a particular environment. This technique is widely used as a biomarker and for microbial ecology studies. It uses a particular kind of RNA known as 16S ribosomal RNA, or 16S rRNA.
Ribosomes are the gene-translating machines in all living things. When a gene on a piece of DNA is copied into a strand of messenger RNA and ferried out of the cell nucleus into the cell fluid, ribosomes there latch onto this mRNA. The ribosomes move along the mRNA strand, reading the code contained in its sequence of nucleotide bases (the As, Gs, Cs and Us, since U replaces T in RNA) and stringing the right amino acids together based on the code to build protein chains.
The genes for ribosomal RNA have changed little over millions of years as organisms evolved. The slight changes that have occurred provide clues as to how closely or distantly various organisms are related.
Because the 16S rRNA gene is very short, just 1,542 nucleotide bases, it can be quickly and cheaply copied and sequenced. So when a scientist has a test tube full of pond water or dirt from an arid mountainside, she must first pull out the rRNA that’s mixed up with all the other RNA, DNA and other stuff in that tube. To do this, she cleans and purifies the sample first, getting rid of unwanted debris.
She then uses one or several techniques designed to break open cells like a kid cracking open a piggybank. Now she has to find the 16S rRNA genes in and amongst all the other genes. Although 16S rRNA genes from different microbes will have a few different nucleotides scattered throughout the sequences, those nucleotides at the very beginning or end of the gene are the same from organism to organism.
The scientist uses several copies of another bit of RNA called a primer. A primer is like a mirror image of a short bit of RNA or single strand of DNA; that is, its sequence of nucleotides is the direct complement to the sequence of nucleotides in a known part of the target RNA or DNA.
In this instance, the primer would be the mirror image of the beginning or end of the 16S rRNA sequence. Because complementary nucleotides pair up like the two halves of Velcro, the primer enables the scientist to pick out the 16S rRNA in the sample. The scientist then uses PCR to make millions of copies of these genes. She then has enough 16S rRNA to compare the sequences of the genes from her sample to libraries of stored 16S rRNA genes from numerous known bacteria.
If some of her gene sequences match up perfectly, she knows that these are microbes that have been previously identified. But if others among her sampled sequences show differences, she knows she has found previously unknown microbes.
The advent of PCR has led to an explosion of microbial gene sequencing in recent years. PCR spells out the entire sequence of the nucleotide bases (the As, Gs, Cs, and Ts) in a DNA molecule that code for a specific protein. Scientists also use sequencing to spell out from start to end every single nucleotide in an organism’s DNA — its entire genome.
Maps of proteins of E.coli (left) and Haemophilus influenzae (right). Courtesy of the National Center for Biotechnology Information of the NIH.
Gene and genome sequencing involve a variety of computers, software programs, automated sequencing machines, fluorescent dyes, lasers, and other tools.
The development of machines that can quickly chop up, separate, realign, and read bits of DNA have greatly speeded up the sequencing process. What used to take a person working by hand to do in a year can now be done by machines in just a few hours. Scientists use gene and genome sequences to precisely compare and differentiate organisms.
If a microbiologist is studying bacteria that bioremediate, or break down, toxic wastes and wants to know which specific genes are active when that bacterium is degrading, say, PCBs, he would likely use a tool called the DNA microarray.
Microarrays enable scientists to monitor the activities of hundreds or thousands of genes at once. All microarrays (also called DNA chips or gene chips) work on the basic principle that complementary nucleotide sequences in DNA (and RNA) match up like the two halves of a piece of Velcro coming together.
Pattern of gene activity on a microarray chip. Courtesy of the National Institute of Allergy and Infectious Diseases.
A microarray consists of an orderly arrangement of bits of genetic material in super-tiny spots laid down in a grid on a suitable surface, often a glass slide with a specially chemically treated surface.
Each spot represents a single gene and contains millions of copies of that gene’s sequence made via PCR. A bacterium’s entire genetic make-up can be contained on a single gene chip. A computer keeps track of which gene is contained in each spot. There can be thousands or tens of thousands of these tiny spots on a single slide.
A specialized robotic machine uses super-thin stainless steel needles to dot the slide with the spots. The robot places the spots at precise intervals as it moves over the surface. The spots are incredibly miniscule, measured in micrometers (millionths of a meter); they typically range from 20 to 100 microns in diameter. Gene chips can be used for a number of purposes, but one of the most common is to determine which genes are expressed or activated under given conditions.
For example, a scientist might wish to figure out which genes in Streptococcus pneumoniae are involved in resistance to an antibiotic. He would first make or purchase a gene chip containing the genes for that bacterium. He would then break open and fish out the RNA from S. pneumoniae grown in plain media and from S. pneumoniae grown in media that contains a low level of the antibiotic (enough to encourage the bacteria to activate resistance genes but not enough to kill all the bacteria).
He would label, then tag the RNA from each sample with a different fluorescent dye. The plain media bacteria could be tagged with a dye that glows green, for example, and the RNA from the antibiotic-tainted media with a dye that glows red. The dye-tagged RNA bits from both bacteria groups would then be washed over the gene chip and left for a period of time to allow complementary strands to link up.
As the bits of RNA randomly bump into the DNA fixed to the chip, RNA sequences that are the complements to fixed DNA sequences latch onto the fixed material. Any unstuck, leftover bits of RNA are then washed off. The scientist uses a laser scanner to detect the fluorescent dyes and create a visual image of the pattern of the dyes.
Some of the gene spots might show up bright green under the scanner, indicating that those genes are active predominantly in bacteria without antibiotic resistance. Others glow yellow, indicating a mix of RNA from both the antibiotic-treated and untreated bacteria latched onto the DNA in those gene spots. This means those genes are active in both antibiotic-resistant and normal bacteria.
Some spots will glow red, however, and these spots indicate genes that are predominantly or only active in antibiotic-resistant bacteria. The brightness of any spot indicates how active those genes are. It’s the genes in the bright red spots that the scientist will now focus on in his efforts to thwart antibiotic resistance.
Because gene chips allow scientists to examine so many genes at once, they have greatly reduced the amount of time it takes to do experiments. Studies that once took months or even years to perform can now be done in a matter of days or even a few hours.
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