Clinical Genomics 101: A Guide to DNA Amplification (PCR)
This is an extraction from our educational e-book, Clinical Genomics 101.
You can download the full guide here.
Each cell contains one complete copy of the host’s genome. For sequencing, however, the best results are obtained when regions are sequenced multiple times, enabling good sequence consensus to be reached during read alignment (which is discussed in chapter 3, in the Clinical Genomics 101). To make this possible and to ensure accurate sequencing, DNA samples are subjected to DNA amplification before they are sequenced.
There are many different techniques for DNA and RNA amplification, such as Loop Mediated Isothermal Amplification (LAMP), Ligase Chain Reaction (LCR), and Nucleic Acid Sequence Based Amplification (NASBA). By far the most common method for DNA and RNA amplification, however, is the Polymerase Chain Reaction (PCR), and so is the method that will be discussed here.
PCR relies on a cycling series of temperature changes which control both the DNA and the enzyme DNA polymerase, which replicates single-stranded DNA (ssDNA) to form double-stranded DNA (dsDNA). In the case of DNA polymerases that require heat activation, the reaction chamber is heated to roughly 35°C for up to 10 minutes. This is known as hot-start PCR, and subsequently follows the same workflow as other PCR reactions. If the DNA polymerase does not require heat activation, the reaction starts with denaturation:
- The reaction chamber is heated to 96°C for 30 seconds. At this temperature, the DNA denatures into its single strand form as the hydrogen bonds between base pairs break. For each double strand, two single strand molecules will be released into solution.
- The temperature is lowered to 50-65°C (depending on the primers used) for roughly 30 seconds. This allows DNA primers to anneal to the end of each single strand. Primers are short ssDNA fragments (around 20 nucleotides long) that complimentary bind the target sequence and enable DNA polymerase to bind the molecule to begin replication. The temperature used for this stage is very important. If it’s too high, then the primers will not anneal to the target and replication will not take place. If it’s too low, then the primers will bind into a non-specific manner, meaning that genomic regions other than the target sequence will be amplified.
- The temperature used for fragment extension is dependent on the DNA polymerase being used. One of the most commonly used polymerases is the Thermus aquaticus (Taq) polymerase, which is usually heated to 72°C for roughly 60 seconds. The polymerase binds to the DNA primer and moves along the single strand in the 5′ to 3′ direction, using free nucleotides in solution to reform a double strand that is identical to the starting molecule. At the end of this elongation process, there will be two complete double strands for each DNA fragment at the beginning of the process.
- The previous three stages are repeated multiple times. Each cycle increases the quantity of the target sequence exponentially (the number of DNA copies for each starting fragment is roughly equal to 2n, where n is the number of cycles).
- Although a final elongation step is optional, it is advisable to hold the mixture at rougly 72°C fir 10 minutes once the final cycle has been completed. This ensures that all ssDNA has been elongated to form dsDNA.
Using modern technology, it is possible to automate the PCR process, so that you can amplify your sequences very easily. PCR does carry the risk of introducing an amplification bias to your DNA mixture, which can influence your results later in your workflow, but the ease and simplicity of the technique have made it very widely used.