PCR Technology - BiopharmaDirect

PCR Technology

Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. There are three main thermal cycling steps in PCR experiment. By adjusting the PCR settings accordingly, researchers can increase yield, enhance specificity and shorten the reaction time. Below are the most common PCR methods and their core advantages.

Hot start PCR

This technique is often used to enhance the specificity of PCR amplification. Modified enzymes such as antibodies, affinity ligands, aptamers, or chemical modifiers are often used to inhibit the activity of DNA polymerase at room temperature. Via modification, the binding capacity between the primer and the template, and between the primer and the primer can be reduced in the PCR system preparation stage, 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.

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 generation of primer dimers and the formation of 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.

Nested PCR

Nested PCR is a modification of 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.

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. In order to determine the shortest extension time without losing 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 thermo-stable are prone to denaturation in this high temperature environment.

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.

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.

PCR of High GC content

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 get "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.

Multiplex PCR

Multiplex PCR can simultaneously amplify multiple different fragments in the same PCR reaction tube. 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.

RT-PCR

RT-PCR was originally designed to determine the sequence of adjacent unknown regions. Nowadays, RT-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 RT-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. 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. 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 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. 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 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.