When we are amplifying qPCR in the laboratory, such kind of situation may offer occur: reagent A is used to amplify system 1 and 2 at the same time, and system 1 performs better in amplification than system 2; However, if the same type of reagent B is used to amplify the two systems, the amplification efficiency of system 2 is better than system1.
Is this just a coincidence or is there a specific rule to follow? Do amplification reagents have different compatibility to different systems? Here, let’s carry out an experiment and dive in! First of all, we will take templates with different GC content as variables, and then use a fluorescent probe, namely Probe-based qPCR method, to detect the amplification product.
So what does different GC content mean? Generally speaking, GC content refers to the ratio of Guanine (G) to Cytosine (C) in nucleic acid sequence, also known as G+C ratio or GC ratio, which can be expressed as: [(the total number of G+C) / (the total number of A+T+C+G)] * 100%. In our experiment, DNA sequences with different GC contents are regarded as amplification templates.
Figure 1. Pairing nature of GC base pairs (image from network, all credits to the owner)
Usually, the GC content of amplification template will be limited at 40%~60%, with an optimum of 45%-55%; GC content out of this range can be a barrier to specific amplification. High GC fragments generally refer to fragments with GC content above 60% in DNA, while low GC fragments refer to fragments with GC content below 40% in DNA.
For systems with different GC contents, we compare 3 kinds of probe qPCR master mix for detection, with 3 replicate wells being tested for each. The average CT value is used as the test indicator to determine the amplification efficiency of assay reagents. Comparing the average CT value with the fluorescence endpoint, the smaller the CT value, the higher the detection efficiency. If the ΔCT value is within ±0.5, the sensitivities will vary little.
※ The results of 2 systems with common GC content(53.5%, 50.6%) are as follows:
Figure 2. Amplification results of 2 systems with common GC content
The results show that the amplification efficiencies of the 3 reagents are not significantly different under 2 systems with common GC content (ΔCT values are within ±0.5 for both comparisons). However, it is clear from amplification plots that the fluorescence endpoint of reagent A is much higher than the other two reagents, indicating that under systems with common GC content, reagent A has a higher compatibility than the other two.
※ The results of 4 systems with high GC content(62.30%, 67%, 68.4%, 63.1%) are as follows:
Figure 3. Amplification results of 4 systems with high GC content
The results illustrate that among the 4 systems with high GC content, amplifications of the 3 reagents vary considerably: compared to the other two amplification reagents, reagent B has a smaller CT value and shows significant difference, while the other two reagents differ little in CT value; in the amplification plot, the fluorescence endpoint of reagent B is also higher than the other two. Therefore, reagent B performs better in the compatibility of systems with high GC content.
※ The results of 4 systems with low GC content(34%, 35.5%, 38.8%, 38.1%) are as follows:
Figure 4. Amplification results of 4 systems with low GC content
The results show that the amplifications of the 3 reagents vary significantly among the 4 systems with low GC content: compared to reagent B, both reagent A and reagent C have advantages of amplification in ΔCT values and fluorescence endpoints of amplification plots, and they have no remarkable difference.
To sum up, the compatibilities of 3 reagents are different in systems with different GC contents. Under the system with common GC content, the detection efficiency of reagent A is better than the other two reagents; under the system with high GC content, the overall detection efficiency of reagent B is the best; while it comes to the system with low GC content, reagent A and reagent C perform better. Hence, reagent B is more compatible with the system with high GC content, just as we mentioned in the beginning of the article — amplification reagents have different compatibility to different systems.
How is this possible?
Here is the reason. As an “obstacle” in the process of amplification, specifically in the three stages of amplification, PCR amplification of high GC content has difficulties in the following 3 aspects:
1. Denaturation: The DNA template melts incompletely at conventional denaturation temperatures.
2. Annealing: The primers cannot bind to the template easily in the process of annealing due to the secondary structure formed by the DNA template.
3. Extension: The extension efficiency of the PCR product declines sharply due to the secondary structure formed by the DNA template[1].
Figure 5. Three stages of hot-start PCR amplification based on enzymatic modified antibody
(image from network, all credits to the owner)
In order to increase the specificity and yield of amplification of high GC target fragments, various optimization methods are adopted. In addition to appropriately increasing the denaturation temperature and adjusting the concentration of key reaction elements such as template, for the amplification reagents themselves, the addition of potentiators (e.g. betaine, dimethyl sulfoxide (DMSO), formamide, glycerol, tween-20, etc.) is an effective way to eliminate complex secondary structures and obtain a large number of amplification products specific for systems with high GC content[2]. However, as the concentration of potentiators cannot be controlled easily, their applications in other systems are likely to cause large amplification inhibition[3], thus giving rise to the result that amplification reagents have different compatibility to different systems.
As a professional product and solution provider in veterinary detection, Vazyme has developed reagents for systems with different GC content based on enzymatic technology innovation and a large number of validation experiments, to help reduce the system development cycle and obtain better performing reagents.
[References]
[1] 张争, 张杨, 徐进, 等. 高GC含量青枯菌aac基因PCR扩增体系的建立与优化[J]. 植物保护, 2008(02): 90-93.
[2] 高梅, 刘树人, 李强, 等. 高GC含量DNA序列PCR扩增的引物设计[J]. 华南国防医学杂志, 2014, 028(007): 633-638.
[3] Zheng Z, Yang Z, Jin X, et al. Establishment and optimization of the PCR system for amplification of aac gene from GC-rich Ralstonia solanacearum[J]. Plant Protection, 2008.
