[0:05]Welcome to applied biological materials PCR video series. In this video, we will introduce the basics of PCR.
[0:16]The polymerase chain reaction, or PCR, is a technology indispensable to genetic and molecular biology.
[0:26]Many of its applications are widely used in the world around us, including DNA sequencing, DNA fingerprinting, forensics, detection of microorganisms, and diagnosis of hereditary disease. PCR allows fast and inexpensive amplification of DNA fragments. Because it is able to generate large quantities of DNA from a small amount of nucleic acid, PCR is also referred to as molecular photocopying. PCR depends on a series of 20 to 40 repeated cycles of DNA replication by a DNA polymerase enzyme. After each cycle, the number of DNA strands is doubled and at the end of a 40 cycle reaction, more than 1 trillion copies are generated from a single copy of a DNA molecule.
[1:16]The PCR process is separated into four steps: initialization, denaturation, annealing, and elongation. In the first initialization step, the reaction is heated to 94 to 96° Celsius for 2 to 10 minutes to activate the DNA polymerase and to denature other contaminants in the mixture. In the case of colony PCR screening, this step also facilitates cell lysis to release DNA and denature other cellular proteins. In the denaturation step, hydrogen bonds between the double stranded DNA are broken by heating the reaction to 94 to 98° Celsius for 20 to 30 seconds. After the DNA strands are separated, primers bind to a complementary sequence in the DNA template to guide DNA polymerase replication. In this step, the reaction temperature is lowered to 50 to 65° Celsius for 20 to 40 seconds to allow for optimal primer annealing. After the primers establish a starting point for the enzyme, DNA polymerase starts to incorporate deoxy nucleotide triphosphates in a 5 prime to 3 prime direction to synthesize a new DNA strand. The temperature and extension time for this elongation step depends on the type of DNA polymerase enzyme and the target amplicon. Commonly used Taq polymerase polymerizes at a speed of 1 to 1.5 kilobases per minute and works ideally at 72 to 78° Celsius. The cycle then repeats from denaturation to elongation 20 to 40 times. At the end of the last cycle, there is a final elongation step that keeps the reaction mixture at 72 to 78° Celsius for 5 to 15 minutes. This ensures that any remaining single stranded DNA is fully extended after the last PCR cycle. A final holding step keeps the PCR at 4 to 15° Celsius for an indefinite time, keeping the products for short-term storage.
[3:27]The most common DNA polymerase enzyme used in PCR is Taq polymerase, a thermostable DNA polymerase that allows multiple cycles of amplification without the addition of new enzyme after each denaturation step. Since the initial discovery of Taq DNA polymerase, many variations of the Taq enzyme have been engineered with different attributes that can be utilized for specific PCR applications. For example, we at applied biological materials, also known as ABM, carry a modified Taq enzyme that has decreased error rates. Also, ABM has engineered a robust Taq enzyme that amplifies DNA directly from blood samples. There are many more engineered Taq enzymes and other DNA polymerases available. To visualize the DNA fragments after amplification by PCR, gel electrophoresis is used. By comparing the gel bands to a molecular weight marker, researchers can estimate the size of the amplified products to know if their desired genes were successfully amplified.
[4:39]Many factors can interfere with a PCR reaction. Some are easy to eliminate, while others are trickier to manipulate and would require experience to tackle. In general, factors that are important in PCR include the nucleotide composition of the DNA template, DNA polymerase choice, buffer components, primer design, additives and inhibitors. Depending on the sequence of the target DNA, different strategies may be needed for a successful PCR. For example, GC rich templates have more hydrogen bonds compared to AT rich templates. This results in incomplete strand separation, which may cause the PCR to fail. In addition, high GC content templates tend to lead to more secondary structures, which can arrest the polymerase leading to premature termination. In these cases, secondary structure destabilizers such as DMSO can be added. At the same time, AT rich templates also require special attention because of their low primer annealing temperatures. Non-specific primer annealing can occur under low annealing temperature, and primer specificity can be retained using a combination of lowered extension temperature and the use of additives such as TMAC. Long templates are difficult to amplify because of their higher likelihood of DNA template being broken or degraded by depurination. Depurination refers to a chemical reaction during which the beta N glycosidic bond in the purine nucleoside is cleaved to release a nucleotide base due to hydrolysis. As Taq DNA polymerase will not extend through a purine position during replication, depurination is an important limiting factor of long template amplification. This can be avoided by using a higher pH reaction buffer and decreasing the denaturation time to limit depurination, or by using a proofreading DNA polymerase. As mentioned earlier, there are many variations of DNA polymerases commercially available, each with different features. Depending on the DNA template, choosing the correct DNA polymerase and pairing it with an appropriate buffer can be critical for a successful reaction. Apart from DNA polymerase choice, having a good pair of primers can also dictate the outcome of a PCR. Some considerations to keep in mind include the length of the primer, the annealing temperature, and the sequence of the primer. For example, the pair of primers should not have complementary sequences, otherwise, they can easily lead to self-dimer or primer-dimer formation and compete with template primer annealing. Many naturally occurring substances such as polysaccharides, tannic acid, and EDTA can also inhibit PCR. Some inhibitors may degrade or modify the DNA template, while others can disturb the annealing of primers to DNA, or alter the DNA polymerase activity. Inhibitors can be removed with sample specific nucleic acid isolation protocols, or sometimes, the use of additives can help in reducing the inhibition. For a complete list of PCR additives and their mechanisms of action, please refer to our knowledge base. A combination of favorable factors contributes to a successful PCR. Taking all these factors into consideration, ABM provides a wide range of DNA polymerases and has formulated optimal PCR MasterMixes for each polymerase. We also offer kits that make DNA amplification from plants, tissue and blood samples easy. Check out our comprehensive list of PCR products in the link provided below. Thank you for watching and make sure you follow our next video on the variations of DNA polymerases. Don't forget to leave your comments and questions below.
