DNA Structure, Replication, and Technology
Topics in Depth
The Theme of Polymerase Chain Reaction in DNA Structure, Replication, and Technology
Don't Stop Repeatin'—Polymerase Chain Reaction
There was once a great episode of Star Trek, where aliens called "tribbles" got on the ship and kept breeding to the point that the whole ship was filled with tribbles. This example is not too dissimilar to how the polymerase chain reaction (or PCR, for us nerds) works. You start off with a few pieces of DNA, and in a few hours, you have thousands of copies of the same piece of DNA.
But, why do scientists need all that DNA? Are they a bunch of eccentrics who drape themselves in strands of DNA, or build imaginary friends out of DNA snowmen? Maybe, maybe not. Most scientists need a lot of DNA to conduct studies on what genetics determine whether or not a person will develop cancer, or to artificially make proteins when formulating vaccines or drugs, or to study what types of organisms live in the sea (many of which will not grow in a lab). PCR amplification of DNA has been a magnificent (yes, we went there) tool in biology to study almost everything we now know today. Most people say "the best thing since sliced bread," but scientists say "the best thing since PCR."
PCR is typically used to amplify a specific gene, or portion of gene, so that we can study the function of that gene or gene region. Primers are used to flank the region you want to amplify. Each primer will amplify the gene sequence on both strands, creating a double-stranded gene product. The PCR process follows 3 steps:
- 95 °C Denaturation step. First, you heat the DNA to a high temperature (95 °C) so that the two strands of genomic DNA, and later PCR DNA, separate.
- Annealing Step (at ~ 50 - 60 °C). Second, you reduce the temperature so that DNA primers bind to either
end of the template that you want to amplify. It is important that you
have two primers, one to bind to each strand of DNA.
- Extension Step. Third, you raise the temperature to about 70 °C to activate a DNA
polymerase and elongate the primer with respect to the template strand.
Each step doubles the amount of DNA copies of your target sequence. You can repeat these three steps, denaturation, annealing, and elongation, 20 - 40 times to generate massive amounts of a specific genetic sequence. If you only have one copy of a gene, and you perform 40 cycles of PCR, you will have 240 copies of that gene, or 1 trillion copies.
There are only a few necessary ingredients for PCR to work:
- Template DNA
- DNA primers
- Deoxynucleotide triphosphates (dNTPs)
- Thermophilic polymerase with a buffer.
You are probably wondering, "What's a thermophilic polymerase?" It is a polymerase that works best at high temperatures, like 70 °C, because it comes from the organism Thermophilis aquaticus, an archaebacteria that lives in deep sea vents whose temperature is normally 80 - 90 °C. Why do you use this polymerase instead of regular DNA polymerase? Well, 40 cycles of heat at 95° for the denaturation step will cause the polymerase to break down and lose activity. You try working in 95 °C heat, and see how well you do.
The dNTPs are basically the nucleotides adenine (ATP), guanine (GTP), cytosine (CTP), and thymine (TTP) that are linked to three phosphates instead of one. A dNTP catalyzes the polymerization reaction by breaking off the other two phosphates and linking in the new base to pair with the template strand of DNA. The template DNA can be either genomic or plasmid DNA, or even another PCR product. It is a small amount of DNA that you want to amplify a specific sequence from.
Sequencing DNA: Checking out Bands
One of the most significant applications of PCR technology is the ability to sequence pieces of DNA. DNA sequence information is important because the sequence of DNA determines the amino acid sequence, which then determines the function of the protein. And, many proteins have regions that are easily distinguishable based on their amino acid sequence. These regions are called domains, and often give a clue as to what the function of the protein is.
Sequencing by PCR is essentially the same as regular PCR. However, sequencing PCR will include dideoxynuclotide triphosphates (ddNTPs). These lack both the 3' and 2' –OH groups of the ribose backbone, which prevents further polymerization of DNA. Therefore, you prematurely terminate DNA replication at a specific site.
The way sequencing initially began was by running four PCRs simultaneously with the addition of ddATP, ddTTP, ddCTP, or ddGTP that were radiolabeled. A person could then look at the size of the bands on a gel and figure out the DNA sequence from what ddNTP was added last. You can figure out the sequence of a DNA strand by writing down the bands in sequence from the bottom to the top of the gel. This process has since been improved by using a mixture of ddNTPs where each base is labeled with a different fluorescent molecule. Therefore, you can look at the sequence based on what fluorescent molecule is added at what position. This is one instance where following a band will not get you into trouble, though this is the closest you come to groupies in science.
Cloning: I Like That! Have Another!
The PCR technique is highly useful in biotechnology (more detail on this can be seen in the biotechnology section), particularly for molecular cloning. Thanks to PCR, we can easily amplify specific genes and put them into plasmids that can then be put into bacteria or eukaryotic cells to make proteins. This process allows researchers to easily study the function of a certain gene. Despite the limited numbers of genes in humans (~ 30,000 genes), scientists only know the function of a small set of them. We would know even fewer of their functions without PCR.
Plasmids are small pieces of DNA, around ~ 1 - 1000 kilobases in length, that bacteria use to share genetic information. Sort of like bacterial email. Bacteria have one chromosome, and it contains only the most essential genes for survival. However, sometimes, bacteria need to adapt to new environments, so they share plasmids to help them survive. Plasmids usually carry antibiotic resistance genes, or other genes that improve the bacterial metabolism, which helps the bacteria survive in a host and often leads to disease in the host.
Scientists working on plasmids one day thought, "Why can't we do that?" and decided to start putting in genes that they are interested in on plasmids. Since then, hundreds of genes have been isolated and studied, and we have gained massive insight into the biology of genes by putting them into plasmids and expressing them. One example would be the green fluorescent protein (GFP) from jellyfish. Scientists extracted that gene from jellyfish DNA, put it into a plasmid, and expressed that gene in different tissues. Studying the function of GFP has allowed us to understand how cellular processes work.
Scientists have made a wide variety of animals that make GFP so that they glow green under UV light. Examples include mice, bunnies, cats, flies, and pigs.
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