In 1983, American biochemist Kary Mullis conceived the idea of in vitro amplification of nucleic acids, and in 1985, he invented the process known as polymerase chain reaction (PCR), giving birth to the PCR technique. In 1988, Saiki et al. successfully completed the automatic amplification of DNA with thermostable DNA polymerase isolated from Thermus aquaticus, namely Taq DNA polymerase, making PCR a convenient and universal molecular biology technology.
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: The optimal temperature of conventional Taq DNA polymerase is 72°C, at which the enzyme activity is at its best, while below which, the enzyme is less active. In addition, DNA polymerase is active at low temperatures, leading to the extension of erroneous primer sequences and the formation of primer dimers. Non-specific amplification may also occur with hot-start high-fidelity enzymes, which is mainly related to PCR conditions (Mg2+, annealing temperature, number of cycles, etc.). Non-specificity may result in low yield of target amplicon, reduced sensitivity of target amplicon, and poor downstream application effect.
Today, we will discuss how to 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.
At the high-temperature denaturation step, samples such as cells or tissues are lysed in specially formulated buffers, and then the DNA is released. Direct PCR simplifies workflow and reduces manipulation steps, thus preventing DNA loss from purification steps. The colony PCR identification adopted in current molecular biology labs represents the most typical application of direct PCR. PCR can be carried out on direct samples following simple treatment or dilution.
Both gradient PCR and touchdown 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 determine the optimal annealing temperature, multiple-tube PCR is performed simultaneously on one PCR instrument (a PCR instrument 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 (e.g., for the temperature range of 50 ~ 60°C, set 6 tubes at 50°C, 52°C, 54°C, 56°C, 58°C, and 60°C, respectively). Finally, the most suitable annealing temperature is determined, and conventional PCR amplification is carried out at this annealing temperature.
He, Lingjuan, et al. “Genetic lineage tracing of resident stem cells by DeaLT”. Nature Protocols. 13.10 (2018):2217-2246.
Many components in the PCR system, such as primers, templates, Mg2+, dNTPs, etc., can lead to inaccurate experimental results. For complex genomic DNA templates, conventional PCR often involves 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 the touchdown PCR (TD-PCR) technique in 1991.
Compared with gradient PCR, touchdown PCR is superior in the following aspects. 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 determined 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 redetermine 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 can affect the amplification results. As the annealing temperature increases, the amplification specificity improves while the amplification efficiency decreases. At the beginning of touchdown PCR, high-temperature amplification is performed 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, the specific amplification product has a geometrical advantage. 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 be up to 15°C, from 5°C 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 determining the optimal Tm value, touchdown PCR can quickly and specifically obtain the target amplified fragment. Many PCR instruments now have programs for setting touchdown PCR, which has been widely used in research.
Although touchdown PCR has 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 also non-specific bands, touchdown PCR can be performed for optimization. But if the main band cannot be seen while only visible non-specific miscellaneous bands, touchdown PCR won’t work well either.
Nested PCR is a variation of PCR that uses two (rather than just a single) pairs of PCR primers to amplify a specific fragment. The first pair of PCR primer amplifies fragment similar to conventional PCR. The second pair of primers, called nested primers (as they lie or are nested within the first fragment), binds inside the first amplicon and specifically amplifies the DNA fragments internal to the initial amplicon.
If the first amplification produces incorrect fragments, the probability is quite low that the region would be amplified in a second round by the second pair of primers. Thus, nested PCRhas high amplification specificity.
Step 1: The first pair of primers shown in green binds to the DNA target template. Due to insufficient specificity, it may also bind to other fragments with similar binding sites and amplify targets.
Step 2: Use the second pair of orange primers to bind to the target fragment amplified in the first step, and then perform the second round of amplification. As these primers are nested within the first PCR product, they make it very unlikely that the non-specifically amplified PCR product would contain binding sites for both pairs of primers. Thereby, the second set of primers cannot amplify non-target fragments. This nested PCR amplification ensures that PCR products from the second round of PCR amplification has little or no contamination from non-specifically amplified PCR products from alternative primer target sequences.