Scientific research dry goods | 10 common strategies for PCR
The PCR experiment includes three main thermal cycling steps. In this process, a variety of necessary reaction components are required. By adjusting the PCR settings accordingly, some special experimental results can be obtained, such as increased yield and enhanced specificity. Or shorten the reaction time.
Hot start PCR
It is often used to enhance the specificity of PCR amplification. This method mainly uses enzyme-modified enzymes such as antibodies, affinity ligands, aptamers or chemical modifiers to inhibit the activity of DNA polymerase at room temperature. This modification reduces the binding capacity of the primer and the template, and the primer and the primer in the preparation stage of the PCR system, thereby avoiding non-specific amplification.
Since the activity of DNA polymerase is inhibited at room temperature, the hot-start technology provides great convenience for preparing multiple PCR reaction systems at room temperature without affecting specificity and amplification ability.
What is hot-start PCR?
After the reaction system is prepared, the enzyme modification is released at high temperature (usually higher than 90 ℃) during the initial heating stage or "hot start" stage of the reaction, so that the DNA polymerase is activated. The specific activation time and temperature depend on the nature of the DNA polymerase and the hot-start modifier. For some DNA polymerases, sometimes the activation and initial denaturation steps can be combined into one step.
DNA polymerase based on antibody hot start technology
Landing PCR
Another way to improve the specificity of the PCR reaction is to adjust the parameters of the PCR cycle. In landing PCR, the annealing temperature of the first few cycles is set a few degrees higher than the maximum melting temperature (Tm) of the primer.
The higher temperature helps to avoid the formation of primer dimers and non-specific primer-template complexes, thus reducing undesirable amplification. Therefore, increasing the annealing temperature at the initial stage of PCR can reduce non-specific PCR products and increase specific amplification.
It should be noted that although a higher annealing temperature can prevent primer dimer formation and non-specific primer binding, it may also aggravate the dissociation of the primer and the target sequence, thereby reducing the PCR yield. To overcome this problem, in the first few cycles, the annealing temperature of each cycle is usually lowered by 1°C to obtain sufficient target amplicons.
Once the annealing temperature reaches or "falls" to the optimal temperature (usually 3-5°C lower than the lowest primer Tm), the annealing temperature is maintained for the rest of the cycle. In this way, during the PCR process, the desired PCR products are selectively increased, while ensuring that little or no non-specific amplification occurs.
Nested PCR
Nested PCR is an evolution of standard PCR, which enhances the specificity of the reaction and the yield of target amplicons. In this method, two pairs of PCR primers need to be designed: one pair (outer primer) flanks the target amplified region, and the other pair (nested primer) corresponds to the DNA region to be amplified.
Among them, the outer primer is used in the first round of PCR to amplify the region containing the extended flanking region. Subsequently, the nested primer is used in the second round of PCR, and the first round of PCR product is used as a template.
If the mismatch of the first pair of primers (outer primers) results in the amplification of non-specific products, the possibility that the same non-specific region will be recognized by the second pair of primers and continue to be amplified is very small, so the amplification of the second pair of primers , The specificity of PCR has been improved. One advantage of performing two rounds of PCR is that it helps to amplify a sufficient amount of product from a limited amount of starting DNA.
Fast PCR
In rapid PCR, faster amplification is accomplished by reducing the time required for the PCR step without affecting the amplification yield and efficiency. Fast cycling conditions are particularly suitable for DNA polymerases with high amplification capacity, which can introduce more nucleotides in each combination.
The elongation time required by the Taq polymerase with high synthesis capacity is only 1/2 to 1/3 of the time required by the Taq polymerase with low synthesis capacity, but it can maintain a high amplification efficiency. In addition, if the annealing and extension temperatures of the primers are almost the same, they can be combined into one step to further shorten the PCR time. This process is also called the two-step PCR method.
When using Taq polymerase with low synthesis capacity, such as Taq polymerase, fast cycling conditions may be suitable for short fragments of <500 bp. Amplifying fragments of this size usually does not require prolonging the polymerization time, so the extension step time in the PCR protocol can be shortened.
In order to determine the shortest extension time without loss of product yield, a series of decreasing extension times (a few seconds) can be used to optimize PCR. Each target fragment and primer pair may have varying results, so it is necessary to optimize the rapid PCR under specific conditions.
Another adjustment method for rapid PCR is to shorten the denaturation time and increase the denaturation temperature to 98°C. When using this strategy, it should be noted that enzymes that are not highly thermostable are prone to denaturation under such high temperature environments.
The reaction parameters used when using low-synthetic DNA polymerases to achieve rapid PCR.
Scientific research dry goods | 10 common strategies for PCR
The use of thermal cyclers and thin-walled PCR tubes that can achieve rapid cycling respectively help to achieve rapid temperature change and efficient heat transfer, which can greatly accelerate PCR.
Direct PCR
Direct PCR refers to the direct amplification of target DNA from the sample without the need for nucleic acid separation and purification. In direct PCR, during the high-temperature denaturation stage, materials such as cells and tissues are lysed in a special buffer to release DNA. Therefore, this method simplifies the experimental process, reduces hands-on time, and can avoid the loss of DNA in the purification step.
Comparison of conventional PCR and direct PCR
It is recommended to use a DNA polymerase with high synthesis ability for direct PCR amplification. Cell debris, proteins, lipids, and polysaccharides are also released into the lysis buffer along with the DNA, and they can inhibit the PCR reaction. The DNA polymerase with high synthesis ability can tolerate such inhibitors, making direct PCR amplification possible. Enzymes with high synthesis ability usually have higher sensitivity, so they can successfully amplify trace amounts of DNA from unpurified samples.
High GC content PCR
DNA templates with high GC content (>65%) are difficult to amplify due to the strong hydrogen bond between G and C bases. The GC-rich sequence also involves secondary structure. Therefore, GC-rich sequences can cause DNA polymerase to "stuck" when amplifying along the template and interfere with DNA synthesis.
In order to amplify fragments with high GC content, the double-stranded template must be dissociated so that the primer can bind to the template and the DNA polymerase can read the sequence. In order to overcome strong GC interactions, the most commonly used method is to use PCR additives such as DMSO or auxiliary solvents to help DNA denaturation. However, these reagents usually lower the Tm of the primer, so the annealing temperature needs to be adjusted accordingly.
The DNA polymerase with high synthesis ability has stronger binding ability with the template, which is conducive to the completion of high GC content PCR. Ultra-high thermostable DNA polymerase is also beneficial for high GC content PCR, because higher denaturation temperature (eg, using 98°C instead of 95°C) may promote double-strand dissociation and PCR amplification.
Multiplex PCR
Multiplex PCR can simultaneously amplify multiple different fragments in the same PCR reaction tube. Multiple PCR means not only saving time, reagents and samples, but also being able to compare multiple amplicons at the same time.
When there are multiple primer pairs in a PCR tube, such as in multiplex PCR, it is not possible to optimize the reaction for only one primer pair or target fragment, but to consider all primers and targets, so non-specific amplification may occur And the efficiency is reduced. Therefore, to minimize mismatches caused by non-specific amplification, primers should be carefully designed.
First, the primer sequence should correspond to its target sequence as much as possible, and the Tm difference of all primers should not exceed 5°C. Before starting multiplex PCR, a single PCR reaction should be used to verify the specificity and amplification efficiency of each primer pair.
In addition, the amplicons should have different sizes so that they can be separated and identified by gel electrophoresis. In addition to primer design and amplicon size, the use of hot-start DNA polymerase and buffers designed for multiplex PCR will also help to obtain successful PCR results and improve reaction specificity.
Although multiplex PCR is often used as end-point PCR, its use in real-time fluorescent quantitative PCR has become more and more popular due to its ability in multiplex labeling and detection. In addition, multiple real-time fluorescent quantitative PCR is also often used for the detection of genetic markers for human identification.
Long fragment PCR
Long-segment PCR usually refers to the amplification of DNA fragments larger than 5kb. Long-segment PCR traditionally uses a mixture of Taq DNA polymerase (for rapid extension) and high-fidelity enzymes (for increased accuracy).
With the invention of high-fidelity DNA polymerases with high synthesis capabilities, it is now possible to achieve more accurate long-segment PCR in a shorter time. By designing a strong DNA binding domain in the DNA polymerase, it can amplify long fragments (for example, >20 kb fragments from gDNA) in a short time and achieve high synthesis ability. In addition, the extremely high fidelity (eg, >100 times the fidelity of Taq polymerase) also helps to ensure a low error rate in the amplification of long fragments.
The high synthesis ability of DNA polymerase can significantly shorten the long-fragment PCR reaction time (in this case, the time is reduced by half), and the high fidelity can reduce the workload of screening clones containing the correct insert.
What is high-fidelity PCR?
When amplifying target fragments >10 kb, the PCR program should be optimized according to the following five key points:
1. Make sure to use high-quality, high-purity DNA samples.
2. If the thermal stability of DNA polymerase is low, more enzymes need to be used to make up for the loss of activity due to prolonged cycle time.
3. Lower the temperature of annealing and extension steps to help primer binding.
4. Properly extend the duration of the PCR step, which is helpful for the complete dissociation of template DNA and the binding of primers.
5. Properly extend the PCR extension time to ensure the full-length replication of the target region.
Reverse PCR
Inverse PCR was originally designed to determine the sequence of adjacent unknown regions. It helps to study the promoter sequence of genes; oncogenic chromosomal rearrangements, such as gene fusion, translocation and transposition; and viral gene integration. This method is called inverse PCR because the primers are designed to extend to both sides rather than toward each other as in conventional PCR. Nowadays, reverse PCR is often used for site-directed mutagenesis to replicate a plasmid with the expected mutation.
In the traditional workflow of studying the unknown sequence of genomic DNA, restriction enzyme digestion and ligation are first performed, then reverse PCR is performed, and the PCR amplicons are then sequenced. For gDNA digestion, a restriction endonuclease must be selected for digestion to obtain fragments of suitable length and capable of self-ligation.
At the same time, the selected restriction enzyme cannot cut the known sequence, so that the ligation occurs between the flanking unknown sequences. Use low-concentration restriction digestion of DNA fragments to optimize the ligation step so that it tends to self-ligate rather than multi-fragment ligation (that is, to form a concatenation).
After completing self-ligation, reverse PCR is initiated from a known region of DNA. The obtained amplicon contains a part of known DNA sequence at each end. Subsequently, these amplicons can be sequenced from the end to detect adjacent regions of the aforementioned known sequences.
Quantitative PCR
The degree of sequence amplification (yield) depends on the initial amount of template. PCR is often used to quantify DNA in a sample. Among them, the most common application is quantification of gene expression. Although the end-point PCR method is feasible, it has a major disadvantage, that is, the yield must be determined by gel electrophoresis, which limits the detection sensitivity.
In addition, quantification is performed at the end of PCR, and the amplification at this time has reached a plateau. Therefore, the intensity of DNA gel staining cannot be linearly correlated with the initial amount of DNA. Nevertheless, if semi-quantitative analysis of gene expression by end-point PCR is performed before reaching the plateau, serially diluted DNA samples can be used as the starting material, or the amplicons of a specified PCR cycle can be collected, and the gene can be estimated based on the intensity of gel staining The amount of expression.
It was not until 1993 that Higuchi et al. reported that the use of fluorescent signals for real-time monitoring of PCR amplification overcomes the limitations of end-point PCR quantification. This technology laid the foundation for what we know today as quantitative PCR (qPCR). In 1997, the first qPCR instrument entered the market, enabling PCR to accurately quantify gene expression and copy number. qPCR relies on real-time monitoring of the fluorescent signal of the target fragment amplified in the exponential phase, which overcomes the shortcomings of end-point PCR quantification. Although qPCR can quantitatively detect relative and absolute gene expression, its detection capabilities limit quantitative performance.
Digital PCR (also known as limiting dilution PCR) developed at the same time as real-time fluorescent quantitative PCR in the 1990s achieved true absolute quantification of DNA samples. In digital PCR, a highly diluted DNA sample is distributed to a multi-compartment chip so that each compartment contains at most one copy of the target. Then, the amplification in each compartment is tested, and a positive or negative result is obtained (1 or 0 template copies, respectively; that is, a "digital" result).
Finally, a statistical model (Poisson distribution) is used to determine the copy number of the sample based on the negative reaction part, without the need to quantify the known sample (standard). In addition to gene expression and copy number quantification, digital PCR is also suitable for applications such as discrimination of low-frequency alleles, virus titration, and absolute quantification of next-generation sequencing libraries. The general workflow of absolute quantification using digital PCR.
In short, the improved PCR protocol and the improved DNA polymerase are aimed at improving the results of PCR amplification. Although the basic concepts of PCR have not changed, new PCR methods will continue to promote and simplify molecular biology research.
