The differences among various PCRS

Article source: IVD Sharing Library

In our daily study and life, we often encounter various terms for PCR, including PCR, qPCR, RT-PCR, RT-qPCR, and Real-Time PCR. Many people often find it difficult to figure out the differences among these types of PCR. Here is a brief introduction to the differences among these types of PCR.

I. PCRisWhat?？

PCR is a polymerase chain reaction（Polymerase Chain ReactionIt is the abbreviation of (), a technology invented by American scientist Kary B. Mullis in 1985 for rapidly amplifying specific DNA fragments in vitro. It can replicate millions of DNA copies within a few hours using an extremely small amount of DNA as a template. Mullis was thus awarded the Nobel Prize in Chemistry in 1993. Since its advent, PCR technology has played a significant role in the fields of biological science, molecular diagnosis, paternity testing, forensic identification, and criminal investigation, and is one of the most important technologies to date. After evolution and innovation, PCR technology has developed for three generations:Ordinary PCR, real-time fluorescence quantitative PCR(qPCR) andDigital PCR（dPCR）。

twoThe principle and steps of PCRA sudden

The basic principle of PCR is similar to the replication of DNA in vivo, and its specificity mainly depends on oligonucleotide primers that are complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denaturation, annealing and elongation.

① Denaturation of template DNA: After the DNA template is heated at a high temperature (around 95℃) for a period of time, it dissociates into single strands to enable binding with primers.

② Annealing (renaturation) of DNA templates and primers: When the temperature is reduced to around 55℃, the primers and the single strands of the template DNA combine with each other according to the principle of base complementary pairing.

③ Primer extension: Then, the temperature is adjusted to around 72℃ (the optimal reaction temperature of DNA polymerase). Under the action of Taq DNA polymerase, the DNA template and primer conjugate, following the principle of base pairing and semi-reserved replication, synthesize a new semi-reserved replication chain complementary to the template DNA strand along the 5'→3' direction. After several cycles of denaturation, annealing and extension, the target gene to be expanded can be amplified and magnified by several million times.

Iii. Classification of PCR

1. Ordinary PCR

Ordinary PCR, also known as the first-generation PCR, can amplify the target gene with an ordinary PCR amplification instrument and qualitatively analyze the product through agarose gel electrophoresis.

2. Quantitative Real-time PCR (qPCR)

Real-time fluorescence quantitative PCR, also known as real-time PCR or the second-generation PCR, refers to the addition of fluorescent dyes or fluorescent groups in the PCR amplification reaction system. During the entire PCR reaction process, fluorescence signals are collected to monitor the changes in the amount of amplification products in each cycle in Real Time. Finally, the samples to be tested were quantitatively analyzed through CT values and standard curves. In the "extension" stage of the "denaturation - annealing - extension" amplification process of the polymerase chain reaction in qPCR, the fluorescence signal of the target gene labeled with a fluorescent probe is collected in real time. Through the relationship among three parameters (fluorescence signal -Ct value - initial concentration of the target gene), the copy number of the target gene or the expression level of the gene is ultimately determined.

Real-time fluorescent quantitative PCR has greatly expanded the research and application of PCR technology in the entire life science field, such as infectious diseases, genetic diseases of tumors, transplantation matching, personalized medication and many other medical fields. Especially in the fields of clinical medical testing and food safety inspection, it has developed rapidly and has become the gold standard for the diagnosis of many pathogenic microorganisms. However, since the absolute quantitative analysis results of quantitative PCR ultimately depend on the Ct value and the standard curve, which is its biggest technical bottleneck, in a certain sense, the so-called "quantification" is only relative. Moreover, under the condition of slight differences in the concentration of low-copy target gene molecular templates, its detection sensitivity, accuracy and resolution are all limited.

There are mainly two types of fluorescence commonly used in qPCR: TaqMan probe method and SYBR Green method.

1.Fluorescence probe method (TaqMan technique)：

TaqMan probeIt is the earliest method used for quantification and also the most commonly used detection method in clinical testing.The Taqman probe is one of the most commonly used hydrolysis probes. At the 5 'end of the probe, there is a fluorescent group, usually FAM. The probe itself is a sequence complementary to the target gene. At the 3' end of the probe, there is a fluorescent quenching group. According to the principle of Forster resonance energy transfer (FRET), when the excitation spectra of the reporter fluorescent group (donor fluorescent molecule) and the quenched fluorescent group (acceptor fluorescent molecule) overlap and are very close (7-10nm), The excitation of donor molecules can induce the fluorescence of recipient molecules, while the self-fluorescence weakens. So when the PCR reaction begins and the probe exists intact in the system, the reporter fluorescent group does not emit fluorescence. During annealing, the primer and the probe bind to the template. In the extension stage, the polymerase continuously synthesizes new strands. Due to the 5 '-3' exonuclease activity of DNA polymerase, when it reaches the probe, the DNA polymerase will hydrolyze the probe off the template. The reporter fluorescent group and the quenched fluorescent group are separated, releasing a fluorescence signal. Due to the one-to-one relationship between the probe and the template, the probe method is superior to the dye method in terms of the accuracy and sensitivity of the experiment.Each time a DNA strand is amplified, a fluorescent molecule is formed. The formation of PCR products is completely synchronous with that of fluorescent molecules. The more PCR products there are, the more fluorescence signals accumulate and the greater the fluorescence intensity.

AdvantagesStrong detection specificity; High sensitivity; Suitable for multiplex qPCR detection; No subsequent processing is required after PCR, saving time and raw material costs.

Disadvantage：Different probes need to be synthesized according to different sequences. Hydrolytic dependence of the probeTaq enzymeThe activity of exonuclease is easily affected by the performance of reagents and enzymes when quantified. Quenching is difficult to be thorough and the background is relatively high. The test results make it difficult to determine the actual amplification characteristics.

2.Fluorescent dye method (SYBR Green：SYBR Green Ⅰ is the most commonly used fluorescent dye in fluorescence quantitative PCR, and it can bind to all double-stranded DNA. In the PCR reaction system, when SYBR Green Ⅰ is added, it will bind to the double-stranded DNA during the process, thereby generating a fluorescence signal. Therefore, all the fluorescence signals emitted during the reaction will be directly proportional to the amount of double-stranded DNA in the reaction, and the fluorescence intensity will also increase with the increase of the product. However, since the dye does not specifically bind to double-stranded DNA, false positive results may occur.

AdvantagesThe price is relatively low. Easy to use; There is no selection for DNA templates, and it has good universality. High detection sensitivity.

Disadvantage：False positive results may occur, and the specificity of the amplification products needs to be identified through melting curve analysis. The reaction system needs to be continuously optimized to reduce non-specific amplification. Not suitable for multiplex qPCR testing.

3. Digital PCR (Dig-PCR, dPCR)

Digital PCR, also known as the third-generation PCR, is absolute quantitative PCR. Unlike traditional techniques, based on the Poisson distribution principle, digital PCR technology dispersions DNA or RNA samples into a large number of micro-reaction units (nanoscale), and then performs single-molecule template PCR amplification, fluorescence detection and statistical analysis on the target sequences within numerous micro-reaction units to achieve absolute quantification. It directly detects the original concentration of nucleic acid in the sample without relying on the standard curve and multiple gradient standards with known concentrations. Due to the superior sensitivity and accuracy of this detection method compared to traditional fluorescence quantitative PCR, digital PCR has rapidly gained widespread attention. Its advantages, especially in the detection of trace nucleic acid samples, rare mutations in complex backgrounds, and the identification of nucleic acid copy number variations and minor differences in gene expression levels, have been widely recognized.

4. 逆转录PCR（ Reverse T ranscription - PCR , RT - PCR）

reverse transcription-PCR, also known as reverse transcription-PCR (RT-PCR), is a widely used variant of polymerase chain reaction (PCR). In RT-PCR, an RNA strand is reverse transcribed into complementary DNA, which is then used as a template for DNA amplification through PCR. Transcribed from a single RNA strand into complementary DNAcDNAIt is called "reverse transcription" and is accomplished by RNA-dependent DNA polymerase (reverse transcriptase). Subsequently, another strand of the DNA passes through the deoxynucleotide primer and the DNA-dependent DThe NA polymerase is completed and doubles with each cycle, which is usually PCR.The RNA used as a template can be total RNA, mRNA or RNA products transcribed in vitro. No matter what kind of RNA is used, the key is to ensure that there is no RNase or genomic DNA contamination in the RNA. RT-PCR technology is highly sensitive and widely applicable. It can be used to detect the expression levels of genes in cells, the content of RNA viruses in cells, and directly clone the cDNA sequences of specific genes. It is generally carried out through one-step or two-step methods. In the one-step method, RT reaction and PCR reaction are conducted in the same test tube. In the two-step method, the two reactions occur separately and in sequence.

5Real-time fluorescence quantitative reverse transcription PCR (Real-time RT-PCR, RT-qPCR)

Real-time RT-PCR is a combination of qPCR and RT-PCR, where "RT" stands for Reverse transcription. Therefore, RT-QPCR is a reverse transcription PCR that integrates fluorescence quantitative technology, that is, it uses mRNA or total RNA as the template. First, reverse transcription was performed to obtain cDNA, and then quantitative detection and analysis were carried out using cDNA as a template through fluorescence quantitative PCR. Because RT-PCR can only be used for qualitative analysis but not for quantitative analysis. Just like RT-PCR, RT-qPCR also has two methods for quantitative analysis of RNA: one-step method and two-step method. Both methods require RNA to be reverse transcribed into cDNA first, and then used as a template for qPCR amplification. However, in the one-step method, RT and qPCR are carried out in the same test tube, while in the two-step method, RT and qPCR are performed separately in sequence.

4. Conclusion

PCR, usually referring to the common PCR (first-generation), uses double-stranded DNA as the template and DNTPS as the substrate for amplification to qualitatively amplify double-stranded DNA.

2. qPCR (Real-time PCR) refers to real-time fluorescent quantitative PCR, which uses DNA as a template and DNTPS as substrates to quantitatively analyze the amplified DNA.

3. dPCR (Digital PCR) uses DNA as a template for PCR amplification, does not rely on ct values or standard curves, and achieves absolute quantification of PCR.

4. RT-PCR, or reverse transcription PCR, uses cDNA, which is reverse transcribed from mRNA, as the template and DNTPS as the substrate to amplify DNA. It is a variant of PCR, and the results can only be qualitative but not quantitative.

5. RT-qPCR, or real-time fluorescence quantitative reverse transcription PCR, is a combination of qPCR and RT-PCR. It involves reverse transcription of total RNA or mRNA into cDNA, which is then used as a template and dNTP as the substrate for quantitative analysis by qPCR.