Improving polymerase processivity is beneficial for amplifying long PCR products (e.g. For example, KAPA2G polymerase has a speed of ~150 nucleotides per second - 3-fold higher than Taq. Some DNA polymerases have been engineered to improve their binding domain, thus making them more stable than conventional Taq. Polymerase processivity is defined as the number of consecutive nucleotides a single enzyme can incorporate before being dislodged from the DNA template.Īt 75☌, native Taq polymerases can typically amplify DNA at a rate of 10–45 nucleotides per second - that’s approximately 2 kilobases per minute! During the initial denaturation step of PCR, the high temperature causes the antibody to dissociate from the polymerase, therefore enabling Taq activation. A hot-start Taq mixture commonly contains a polymerase that is bound to an antibody, which inhibits the activity of Taq at temperatures lower than 90☌. One way to increase the specificity of DNA polymerase and the PCR is to use a hot-start Taq. For example, mispriming and premature target amplification of undesired products can sometimes occur during the reaction set-up at room temperatures. This broad temperature activity profile can actually hinder the specificity of the PCR. When Taq polymerase was first extracted and analyzed by Alice Chien and colleagues in 1976, it was noted that the enzyme is active across a range of temperatures (~40–80☌). Both DNA polymerase and the PCR primers contribute to the overall specificity of the reaction. Specificity of the PCR is measured by non-specific amplification of unintended targets. For example, the half-life of Taq polymerase at 95☌ is 40 minutes, whereas the half-life of the hyperthermophilic Deep Vent DNA polymerase extracted from the Pyrococcus species GB-D is several hours at 98–100☌. There have been various thermostable polymerases identified to date, each with its optimal temperature for activity and a unique half-life profile at temperatures greater than 95☌. The thermostability of DNA polymerases is defined by how long they remain active at the extreme range of temperatures used in PCR. In some cases, it may be more appropriate to use a mixture of different polymerases to optimize the thermostability, specificity, processivity, and fidelity requirements. It’s therefore important to consider the PCR application with the 4 key attributes of the polymerase: There are various types of DNA polymerase commercially available, each with their own unique properties. For example, gDNA contains more bases and hydrogen bonds compared with cDNA therefore gDNA requires a longer initial denaturation time to ensure the double-stranded DNA is dissociated. The larger the DNA template, the longer the initial denaturation phase is. The high temperature denatures hydrogen bonds that hold the complementary bases together, therefore forming two single strands of DNA. Step 1: Initial Denaturationĭouble-stranded DNA is first heated to around 94☌ for a few minutes. The denaturation, annealing, and extension (steps 2–4) steps form the PCR cycle, which is repeated multiple times to increase the amount of final PCR product. How does PCR work?Ī typical PCR is composed of the following steps: The PCR reaction buffer contains essential elements required for a successful reaction including magnesium ions (Mg2+), which is a cofactor for DNA polymerase. To create new strands of DNA during the PCR there must be available deoxyribonucleoside triphosphate (dNTP) bases - adenine, thymine, cytosine and guanine. You can learn more about the attributes of DNA polymerases below. Named after the thermophilic bacteria Thermus aquaticus from which it was originally extracted, Taq polymerase can work efficiently at high temperatures. The type of polymerase commonly utilized in PCR is known as Taq DNA polymerase. Once bound to target regions, PCR primers enable DNA polymerase to attach and extend the new sequence. These primers anneal to complementary regions of single-stranded DNA. PCR primers are designed as pairs, referred to as forward and reverse primers. The source of DNA can include genomic DNA (gDNA), complementary DNA (cDNA) or plasmids. DNA, either single or double-stranded, is the standard template for PCR.
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