Since the invention of polymerase chain reaction (PCR) by Kary Mullis in 1983, the technology has played an important role in the field of life sciences, for which Mullis won the Nobel Prize in Chemistry in 19931. In 1988, Saiki et al. successfully completed the automatic amplification of DNA with a thermostable DNA polymerase isolated from Thermus aquaticm, namely Taq DNA polymerase, making PCR a convenient and universal molecular biology technology2.
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However, with the wide application of conventional PCR technology in various fields of molecular biology, phenomena such as small sample size, precious samples, and non-specific amplification often occur.
Non-specific amplification can be caused by:
1. The optimum temperature of common Taq DNA polymerase is 72℃, and the activity of the enzyme is the best at this time. Below 72℃, the enzyme is less active, so the DNA polymerase is active at low temperature resulting in misguided target extension and primer dimer formation.
2. Non-specific amplification may also occur when hot-starting high-fidelity enzymes, which is mainly related to PCR conditions (Mg2+, annealing temperature, number of cycles, etc.).
Non-specificity will result in low yield of target amplicon; reduced sensitivity of target amplicon; poor downstream application effect. Today we will talk about how to successfully amplify the target fragments we need.
Direct PCR refers to the amplification of target DNA directly from a sample without nucleic acid isolation and purification. In direct PCR, samples such as cells or tissues are lysed in specially formulated buffers. During the subsequent high temperature denaturation step, the DNA is released. Direct PCR simplifies workflow and reduces manipulation steps, thus preventing DNA loss from purification steps.
Figure 1. Workflow of direct PCR
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Both Touchdown PCR and gradient PCR optimize the annealing temperature in the reaction system, but the principles are different.
Gradient PCR means that when the annealing temperature is not very clear, in order to find the optimal annealing temperature, multiple-tube PCR is performed simultaneously on one PCR machine (a PCR machine that supports setting the gradient annealing temperature is required). Each tube is placed on a different column or row in the instrument, and PCR is performed separately. Finally, the most suitable annealing temperature is found, and ordinary PCR amplification is carried out at this annealing temperature.
2. Gradient PCR reaction distribution3
Many components in the PCR reaction, such as primers, templates, Mg2+, dNTPs, etc., can lead to inaccurate experimental results. For complex genomic DNA templates, ordinary PCR often has non-specific amplification, and the desired ideal product cannot be obtained. In order to solve the problem of non-specific amplification of PCR, Don et al. invented touchdown PCR (TD-PCR) technology in 19914.
Compared with gradient PCR, touchdown PCR has advantages. First, multiple reactions or multiple tube reactions are required to select an appropriate annealing temperature for gradient PCR. Second, even if the optimal annealing temperature is found through multiple experiments, the optimal annealing temperature may change when the same amplification is performed by replacing other PCR instruments. At this time, it is necessary to rediscover the optimal annealing temperature. However, touchdown PCR can obtain a good amplification effect with only one reaction, avoiding the optimization and determination of the optimal renaturation temperature for each pair of primers. In addition, touchdown PCR largely weakens the restriction of instrument performance on the amplification effect.
The annealing temperature in the PCR reaction will affect the amplification results. As the annealing temperature increases, the amplification specificity becomes better, while the amplification efficiency becomes lower. At the beginning of touchdown PCR, high temperature amplification is used to obtain specific amplification products. After the abundance of the target gene increases, lowering the amplification temperature can improve the amplification efficiency. When the annealing temperature is lowered to the level at which non-specific amplification occurs, there is a geometrical advantage of the specific product. Non-specific sites in the remaining reactions cannot compete with specific sites due to their low abundance, resulting in a single dominant amplification product.
Annealing temperature setting
Typically, the annealing temperature range for touchdown PCR can span 15°C, from a few degrees Celsius above the Tm value to around 10°C below it. Cycle 1-2 times at each temperature, then cycle 10 times at the lower annealing temperature.
Touchdown PCR is suitable for experiments where primers are changed frequently. Without knowing the Tm value and without the trouble of finding the best Tm value, touchdown PCR can quickly and specifically obtain the target amplified fragment. Many PCR machines now have programs for setting touchdown PCR, and it has been widely used in the research field.
Although touchdown PCR has certain advantages, it is not a panacea. Touchdown PCR can only be “icing on the cake”, that is, if the main band can be seen, but there are stray bands, touchdown PCR can be used for optimization. But if you can’t even see the main band, only the stray band, then using touchdown PCR won’t work well either.
Figure 3. The program of touchdown PCR
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Nested PCR is a variant of PCR that uses two pairs of PCR primers to amplify intact fragments. The first pair of PCR primers amplifies fragments similar to common PCR. The second pair of primers, called nested primers, bind to the first PCR product and specifically amplify the DNA fragments located in the first PCR product.
If the first amplification produces the wrong fragment, the probability of primer pairing and amplification of the wrong fragment is very low. Therefore, nested PCR amplification is specific.
1. The first pair of yellow primers binds to the DNA template. Due to insufficient specificity, it may also bind to other fragments with similar binding sites and amplify the product.
2. Use the second pair of black primers to bind to the target fragment amplified in the first step for the second round of amplification. Since the second pair of primers is located inside the PCR product of the first step, it is extremely unlikely that the non-target fragments contain two sets of primer binding sites, so it is impossible for the second set of primers to amplify the non-target fragments. This nested PCR amplification ensures that the second-round PCR products are little or completely free from contamination by non-specific amplification caused by poor primer pairing specificity.
Figure 4. Nested PCR
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1. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K., Hoorn, G. T., & Arnheim, N. (1985). Polymerase chain reaction. Science, 230, 1350-1354.
2. Lawyer, F. C., Stoffel, S., Saiki, R. K., Chang, S. Y., Landre, P. A., Abramson, R. D., & Gelfand, D. H. (1993). High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′to 3′exonuclease activity. Genome research, 2(4), 275-287.
3. He, L., Li, Y., Huang, X., Li, Y., Pu, W., Tian, X., … & Zhou, B. (2018). Genetic lineage tracing of resident stem cells by DeaLT. Nature Protocols, 13(10), 2217-2246.
4. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., & Mattick, J. S. (1991). ‘Touchdown’PCR to circumvent spurious priming during gene amplification. Nucleic acids research, 19(14), 4008.