4 Experiment (Lab Period 4) Amplification of a specific DNA fragment by the Polymerase Chain Reaction (PCR) Once DNA is extracted from an organism, many different types of analyses can be performed. Biologists are often interested in studying a specific gene or a specific chromosome region to determine how that gene functions at the molecular level. To do this that region containing the gene of interest must be isolated away from the rest of the DNA. Typically, a gene you want to study is composed of 1000 nucleotides of DNA (or less) while most organisms have on the order of 107 to 109 nucleotides of DNA in each cell. Therefore, unless you are interested in studying the entire DNA complement of an organism, it becomes necessary to go through several steps following DNA extraction to isolate that small fragment of DNA that you actually want to study. The isolation of a specific DNA fragment is frequently achieved by cloning. This is a complex and time-consuming process. To clone a fragment of DNA a biologist has to essentially cut the DNA into many small pieces and sort through all of the pieces to find the one containing the gene in which he or she is interested. In recent years the need to clone a fragment of DNA in every situation has been eliminated by the development of the Polymerase Chain Reaction (PCR) by Kerry Mullis. The advantage of PCR over cloning is that, as we will see below, you can specifically target the gene you want and make many copies of it in a test tube. This means that you do not have to sort through all of the pieces of DNA, you simply target the one you want. Of course you need to know something about the gene from previous work in order to be able to target it specifically, but with recent advances in genome sequencing this information is becoming readily available for most genes. PCR has become widespread since it is much quicker than cloning, and much cheaper. The idea behind PCR is that you exploit the natural system of DNA replication. Every cell makes a copy of its DNA before it divide, a reaction which is performed by an enzyme called DNA polymerase (along with a lot of other proteins). During replication the two strands of the DNA molecule are separated and each strand is used as a template to make a new strand. The result is that two identical new strands of DNA are generated from one original copy. This process cannot start from scratch: instead, it must be `primed'. What this means is that DNA polymerase cannot simply start making a new strand of DNA on a template but, rather, can only extend an existing second strand. Furthermore, the enzyme can only do this by adding nucleotides to the 3' end of the new strand, it cannot add nucleotides to the 5'. Organisms get around this by generating short pieces of RNA on the template using an enzyme called a primase. This short piece of RNA (a primer) can then be extended by DNA polymerase and, later, replaced by DNA. PCR uses this system of DNA replication by getting DNA polymerase to replicate, many times over, a specific region of the chromosome. Since DNA polymerase needs a primer you will control the region of replication by providing the primers. In PCR, many copies of two short
pieces of single stranded DNA act as primers and are added to the DNA you have extracted from the cell (the template DNA) along with DNA polymerase and a mixture of the four nucleotides (dATP, dCTP, dGTP and dTTP). The mixture is then heated to almost 100C during which time the DNA that you extracted denatures (i.e. the two strands separate). The mixture is then allowed to cool so that double stranded DNA reforms. However, you have added a lot of copies of the short pieces of DNA so that as double stranded DNA forms some of these short pieces will bind to the template DNA where they are complementary. At point this the DNA polymerase you have added can extend this primer using the nucleotides in the mixture and you have a new copy of a specific area of the chromosome. By adding two primers, one that is complementary to each end of the gene you want to study, you can target the gene by PCR. One primer is complementary to one strand of the template at one end of the gene and the second is complementary to the other strand at the other end of the gene. Using this design you can then perform multiple steps of the reaction described above. This reaction consists of 3 steps that together are referred to as a cycle: 1 Heating (denaturation). This is usually achieved by 30 seconds at 95C. 2 Cooling (renaturation). The temperature of annealing varies between 40 and 60C. 3 Replication. This is typically from 1 to 3 minutes at 72C. The cycle is repeated 30 to 40 times in a single PCR. During each cycle, the region between the two primers is used as a template to make new copies. All of the new copies become templates for subsequent cycles so that each cycle doubles the number of copies of DNA in the region between the two primers. After 30 cycles you get, in theory, 230 copies of the gene you want to study (although it is not usually the case that you get a doubling every single cycle). This is commonly referred to as `amplification'. This means that you need not sort through all of the DNA anymore. Although there is original genomic DNA still in the reaction it is swamped out by these new copies that can easily be isolated and studied.
Procedure 1. Label 2 different 500? l centrifuge tubes as C1 and R1 and include your group number on each. The first will be your control and the second your reaction. (The control will have all of the components of PCR except the template DNA and this acts as a negative control. PCR is very sensitive in that a single template molecule is sufficient to result in a product. Therefore, it is critical to ensure that the template you add is the only template available for reaction. If there are any contaminating DNA molecules in any of the other reagents that can cause a reaction we will see this in the negative control. If you observe a product in the negative control then you cannot use the results of your reaction. On the other hand, if, as expected, you observe no product in the negative control but a product in the reaction tube then you can be confident that the reaction amplified the template you wanted.) 2. To both tubes add: i. 24 ? l primer mix. This contains both primers. ii. 8 ? l dNTP mix. iii. 8 ? l buffer mix 3. To the tube C1 add 5 ? l water. 4. To the tube R1 add 5 ? l of Template. 5. Add 100? l mineral oil to both C1 and R1. Be careful when you pipet mineral oil, you will find that it acts very differently than the substances you have pipeted up until now. The mineral oil is inert (it doesn't contribute to the reaction) but it prevents evaporation of your sample as it goes through successive stages of 95C. 6. Place both tubes in the temperature cycler. When all groups are ready the temperature cycles will begin. The purpose of the cycler is to control the temperature so that the reaction goes through the 3 stages outlined above. The reaction we will use consists of 35 cycles of: 1 30 seconds at 95C. 2 1.5 minutes 42C. 3 1.5 minutes at 72C. Before these cycles begin the temperature cycler bringing the tubes to 95C for 30 seconds and then to 80C for 10 minutes. The first step destroys any contaminating proteins that may interfere with the reaction. When the tubes are at 80C then you add 5 ? l Diluted Taq DNA Polymerase to both C1 and R1. The tubes will be left to go through the cycles and be ready for analysis next week. Analysis: The results of your PCR experiment will be analyzed by gel electrophoresis in experiment 5.