Research progress and application of real-time fluorescent quantitative PCR

Research progress and application of real-time fluorescent quantitative PCR

(Zhang Wei, Shen Lisong Experimental Diagnostic Center, Shanghai Children's Medical Center, Xinhua Hospital, Shanghai Second Medical University)

Abstract: The rapid development of polymerase chain reaction has made it an important tool in molecular biology laboratory. Real-time quantitative PCR (PCR) technology has high sensitivity, good repeatability, fast speed and less pollution. Another step forward. This paper introduces the principle of real-timeQ-PCR technology, five main fluorescent chemistry and quantitative methods, and also discusses the shortcomings of this technology, and summarizes its main applications in the research.

The polymerase chain reaction (PCR) technology has been invented for nearly 20 years. During this period, its technology has been continuously developed. The real-time quantitative PCR technology in recent years has realized the qualitative to quantitative PCR. Leap, it has become an important tool in molecular biology research because of its strong specificity, high sensitivity, good repeatability, accurate quantitative, fast speed and fully enclosed reaction. This paper reviews the technology and its application.

1 Methodology of real-time PCR technology

1.1 Principle The so-called real-timeQ-PCR technology refers to the addition of fluorophores in the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR in real time.

Cheng, finally the method of quantitative analysis of unknown templates by standard curve. In the development of real-time technology, two important discoveries play a key role: (1) the discovery of the 5' exonuclease activity of Taq DNA polymerase in the early 1990s, which can degrade specific fluorescent labeling The needle thus makes it possible to indirectly detect PCR products. (2) The use of the fluorescent double-labeled probe thereafter allows the entire reaction process to be monitored in real time in a closed reaction tube. The combination of these two discoveries and the commercialization of the corresponding instruments and reagents have led to the use of real-time PCR methods in research work.

The copy number of DNA generated during the PCR reaction increases exponentially. As the number of reaction cycles increases, the final PCR reaction no longer generates an exponential template and enters the plateau. In conventional PCR, gel electrophoresis is used to separate and fluorescent staining is used to detect the final amplification product of the PCR reaction, so the quantification of the PCR product by this endpoint method is unreliable. In real-timeQ-PCR, the entire PCR reaction amplification process is monitored in real time and the fluorescence signal associated with the amplification is continuously analyzed. As the reaction time progresses, the detected fluorescence signal changes can be plotted as a curve. . In the early stages of the PCR reaction, the level of fluorescence produced cannot be clearly distinguished from the background, and the generation of post-fluorescence enters the exponential phase, the linear phase, and the final plateau, so that the amount of PCR product can be detected at some point in the exponential phase of the PCR reaction. And thereby inferring the most templates

Initial content. In order to facilitate comparison of the detected samples, in the exponential phase of the real-time Q-PCR reaction, the domain value of a fluorescent signal is first set. Generally, this threshold is the fluorescent signal of the first 15 cycles of the PCR reaction. As a fluorescent background, the default setting for the fluorescence domain value is 10 times the standard deviation of the 3-15 cycles of the fluorescent signal. If the detected fluorescent signal exceeds the domain value and is considered to be a true signal, it can be used to define the number of domain value cycles of the sample (the meaning of the CO.Ct value is: when the fluorescent signal in each reaction tube reaches the set domain value) The number of cycles experienced. Studies have shown that there is a linear relationship between the Ct value of each template and the logarithm of the starting copy number of the template. The more the starting copy number, the smaller the Ct value. The standard for using the known starting copy number The product can make a standard curve, so as long as the Ct value of the unknown sample is obtained, the starting copy number of the sample can be calculated from the standard curve.

1.2 Fluorescence Chemistry

At present, there are five main fluorescent chemical methods used in real-time Q-PCR: DNA binding staining, hydrolysis probe, molecular beacon, fluorescent labeling primer, and hybridization probe. They can be divided into two major categories: amplified sequence specific and non-specific detection.

The basis for non-specific detection of amplified sequences is DNA-bound fluorescent molecules such as SYBRgreen1 [33 and other fluorescent dyes. In the early development of Real-timeQ-PCR, the simplest method was used. In the PCR reaction system, an excessive amount of SYBRgreen1 fluorescent dye was added, and the SYBRgreen1 fluorescent dye was specifically incorporated into the DNA double strand to emit a fluorescent signal. The advantage of fluorescent dyes is that it can monitor the amplification of any dsDNA sequence, does not require the design of the probe, makes the detection method simple, and reduces the cost of detection. However, because fluorescent dyes bind to any dsDNA, they can also bind to non-specific dsDNA (such as primer dimers), making the experiment prone to false positive signals. The problem of primer dimers can now be solved with software with melting curve analysis.

The amplification sequence-specific detection method is to detect the product by using a tomb-specific oligonucleotide probe labeled with a fluorescent dye in a PCR reaction, which can be further divided into a direct method and an indirect method. The indirect method is to use the strategy of hydrolysis probes. The most widely used TagMan system in real-time Q-PCR is the use of this principle. During PCR amplification, a specific fluorescent probe is added at the same time as adding a pair of primers. The probe is an oligonucleotide, and a reporter fluorophore and a quenching fluorophore are respectively labeled at both ends. The end-fluorescent tomb mass absorbs energy and transfers the energy to the adjacent 3'-end fluorescence quenching tomb (fluorescence resonance energy transfer, FRET). Therefore, when the probe is intact, the 5'-end fluorescent tomb of the probe is not detected. Fluorescence emitted. However, in PCR amplification, when the template in the solution is denatured and then annealed at a low temperature, the primer and the probe are simultaneously bound to the template. Mediated by primers, extending forward along the template to the probe junction, strand displacement occurs, 5'-3' exonuclease activity of Taq enzyme (this activity is double-strand specific, free single strand The probe is unaffected) the fluorophore attached to the 5' end of the probe is cleaved from the probe, freed from the reaction system, thereby being detached from the shielding of the 3, terminal fluorescence quenching group, and receiving a fluorescent signal to emit a fluorescent signal. That is, each time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.

The direct method means that the fluorescently labeled probe directly combines with the amplification product to produce fluorescence directly. The molecular beacon ['] belongs to this category. It is essentially a fluorescent hairpin probe. When the probe molecule is in a hairpin structure, the fluorophore binding at its two ends is close in distance. So that an energy transfer effect occurs without fluorescence. When the complementary sequence occurs, the probe hybridizes with the DNA, and the probe transforms into an open structure, which is linear. The reporter fluorophore and the quenching fluorescent tomb are spatially separated from each other, and the fluorophore is detached from the quenching. The influence of the tomb, resulting in fluorescence that can be detected. Fluorescently labeled primers are a joint molecular probe system derived from the conceptual change of molecular beacons. They combine the sequence of the fluorophore-labeled hairpin structure directly with the PCR primers, thereby allowing the fluorescent labeling group to be directly incorporated into the PCR. In the amplification product. There are currently two main types: sunriseprimes and scorpionprimes. Hybridization probes use two specific probes, in which the 3' end of the upstream probe is labeled with a donor luciferin, and the 5' end of the downstream probe is labeled with a receptor fluorescein (acceptor) ). In the template annealing phase of PCR, the two probes simultaneously hybridize with the amplified product and form a head-to-tail binding form, so that the distance between the donor and the acceptor fluorescein is very close, and the two produce fluorescence resonance energy f transfer (FRET). In contrast to the manner described above for hydrolyzing probes, the acceptor fluorescent tombs fluoresce; when the two probes are in a free state, no fluorescence is produced. Since the two probes are used in the reaction, the specificity of the method is increased, and the sequence of the binding to the oligonucleotide probe can also be analyzed by using a melting curve to obtain useful information. information.

1.3 quantitative method

In real-timeQ-PCR, there are two strategies for template quantification: relative t and absolute. Relative t is the change in f of a target sequence relative to another reference sample in a given sample. Absolutely t refers to the use of known standard curves to estimate the to sample of an unknown

1.3.1 Relative curve of the standard curve method Since the expression of f is officially relative to the f of a certain reference in this method, the standard curve of the relative f is easier to prepare, and it is only necessary to know the standard used. Its relative dilution can be. The t of the target sequence of the sample from the entire experiment is from the standard curve, and finally must be divided by the reference t, ie the sample with the reference being 1, and the other samples being n times the reference t. In order to standardize the RNA or DNA added to the reaction system in the experiment, an endogenous control substance is often amplified simultaneously in the reaction. For example, in the study of tomb expression, endogenous control substances are often some of the tombs of the house (such as beta-actin). , glycerol-3-phosphate deaminase UAPDH, etc.).

1.3.2 Comparison of the relative quantitative comparison of the CT method The difference between the CT method and the standard curve method is that it uses a mathematical formula to calculate the relative t, provided that each cycle doubles the number of products I, The exponential phase of the PCR reaction yields the CT value to reflect the amount of the starting template. The difference in one cycle (CT = 1) is equivalent to a difference of 2 times the number of the starting board. However, this method is based on the premise that the target tomb cause and the amplification efficiency of the endogenous control substance are consistent, and the offset of the efficiency will affect the estimation of the actual copy number.

1.3.3 Absolute quantification of the standard curve method The difference between this method and the standard curve method is that the amount of the standard is known in advance. Plasmid DNA and RNA transferred in vitro are often used as preparations for the absolute fi standard. The f of the standard can be determined based on the absorbance value at 260 nm and converted to its copy number using the molecular weight of DNA or RNA.

2 application of real-time fluorescent quantitative PCR technology

Real-timeqPCR is used in a wide range of applications, including mRNA expression studies, DNA copy number detection, and single nucleotide polymorphisms (SNPs). The following is an overview of the current real-timeQ-PCR in the detection of translocation tombs, cytokine expression analysis, tumor resistance tocstone expression studies, and quantitative monitoring of viral infections.

2.1 Detection of minimal residual disease Tumor diseases, especially malignant tumors of the blood

Often accompanied by a translocation of a specific gene, this translocation can often be used as a tumor marker to monitor the clinical treatment effect. Although improvements in treatment regimens over the past few decades have greatly extended patient survival, patients in remission have a risk of recurrence. Therefore, the detection of minimal residual disease (MRD) is critical to further adjustment of the treatment regimen. The application of Real-timeQ-PCR is becoming an indispensable research tool for detecting micro-residue molecular markers of tumors. The quantitative measurement of tumor fusion genes can guide clinical individualized treatment of patients. The most common chromosomal abnormality in acute myeloid leukemia (AML) is the translocation t(8;21) (g22;q22), in which the AML-1 transcription factor gene and

Fusion of the MTG8 gene on chromosome 8 causes normal transcriptional regulation of AML-1 to be affected, which may be the cause of leukemia. The current study [1o] demonstrates that the use of real-timeqPCR to detect fusion genes can help quantify MRD in these patients, which is valuable as an indicator of prognosis or assessment of treatment options. The same method was also used to quantify other translocation fusion gene levels, such as BCR-ABI_fusion tomb in chronic myelogenous leukemia (CML) [" leukemia-specific TEL of acute lymphoid cell leukemia (ALL) - AML1 fusion gene [iz], chromosomal translocation t (14; 18) (g32; g21) [Is" and bel-2 rearrangement C14] of follicular lymphoid tumor (Fl,). Many studies have benefited greatly from the application of the real-time Q-PCR method, and the use of Real-time Q-PCR will continue to expand as technology advances.

2.2 Analysis of cytokine expression Cytokines are regulatory proteins that play a central role in the immune system by modulating immune responses, including lymphocyte activation, proliferation, differentiation, survival, and apoptosis. Many different types of cells secrete this low-molecular-f protein, including lymphocytes, antigen-presenting cells, monocytes, endothelial cells, and fibroblasts. Cytokines can be divided into different groups: interleukin (II.1-"IL-23), interferon (IFN-+, IFN-Y, etc.), colony stimulating factor (CSF), tumor necrosis factor (TNF), tumor Growth factors (TGF-month, etc.) and chemical factors (MCP-1, MIP-1, etc.) [I]. To elucidate the immunopathogenic pathways in many inflammatory responses, autoimmune diseases and organ transplant rejection, cytokines

The reliable determination of the mRNA expression profile is important. Although the cytokine t content in the sample to be tested is often extremely low, real-time reverse transcription PCR (RT-PCR) is increasingly favored in the cytokine determination with its high sensitivity and accuracy.

2.3 Tumor resistance is a major obstacle to the treatment of cancer patients. Since drug resistance limits the successful treatment of many tumors, it is important to study the drug resistance mechanisms of tumor cells. The main mechanisms of drug resistance found in the current study are: ATP-binding cassette atomic superfamily (ATP-bindingcassette superfamily) membrane transporter-mediated resistance [15], these proteins include: MDR-1 tomb encoded P-gun Protein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-associated protein (LRP), breast cancer resistance protein (BCRP), etc., enzyme-mediated resistance, including topoisomerase (Topo) ), Glutinous Glucosamine (GSH) and Glutinous Ginseng-S-transfer sputum (GST), protein kinase C (PKC), deoxycytidine kinase, etc.; apoptosis-mediated tolerance Drug ["], such as bcl-2 family, p53 tomb, c-myc, etc. Multidrug resistance (MDR) is the result of multiple factors and multiple mechanisms. Real-time reverse transcription PCR (RT-PCR) It is a useful means to understand tumor resistance and guide clinical treatment strategies. It can observe changes in the expression of drug-resistant tombstone mRNA in tumor cells before and after treatment, so as to timely adjust the treatment plan and evaluate the prognosis of the disease.

2.4 The development of viral infections The development of amplification techniques has led to an increase in the ability to qualitatively or tune the detection of viruses, and has also made it possible to study the relationship between viral load and disease progression. Real-timeQ-PCR is an amplification technology mainly used in scientific research and diagnostics. It can not only characterize viruses, but also because of its small inter-assay and intra-assay differences and good repeatability, so it can be convenient, fast and sensitive. Accurately quantify the sequence of viral DNA or RNA, and more importantly, dynamically study the reactivation or persistence of potential viruses throughout the disease, allowing clinicians and virologists to detect clinical changes, such as antiviral therapy. The effect, the emergence of drug resistance mutations, etc. At present, it is more commonly used to quantitatively measure CMV infection in patients who use immunosuppressants in organ transplantation using Real-time Q-PCR. Studies have shown that detection of CMV infection in patients with bone marrow transplantation is more sensitive than traditional pp65 antigen test, anti-CMV drug treatment can reduce the virus content in blood, Real-timeQ-PCR for rapid quantitative bone transplantation patients CMV infection and monitoring CMV resurrection is a useful tool.

3 existing problems and application prospects

In real-time PCR, there are still some problems in PCR quantification methods, whether it is relative quantification or standard curve quantification. The preparation of standards is an indispensable process in the quantification of standard curves. At present, because there is no uniform standard, the samples used to generate the standard curve used by each laboratory are different, resulting in a lack of comparability of the experimental results. In addition, when using eal-timeqPCR to study mRNA, it is limited by the different reverse transcription (RT) efficiency of different RNA samples. In the relative quantification, the premise is that if the endogenous control is not affected by the experimental conditions, it is also the key to the reliableness of the experimental results to reasonably select appropriate endogenous controls that are not affected by the experimental conditions. In addition, compared with the traditional PCR technology, the shortcomings of Real-timeQ-PCR are [18]: (1) due to the use of closed detection, the detection step of post-amplification electrophoresis is reduced, so it is impossible to monitor the expansion. The size of the product is increased; (2) because of the type of fluorescein and the limitations of the detection source, which limits the application ability of the composite multiplex detection of real-timeQ-PCR; (3) the cost of real-timeQ-PCR experiment It is relatively high, which limits its wide range of applications. With the continuous improvement and development of technology, real-timeQPCR has become the main tool of scientific research. The future application prospect of this technology is encouraging. On the one hand, real-timeQ-PCR technology combined with other molecular biology techniques makes the quantitative pole A small amount of gene expression or DNA copy number is possible. On the other hand, the development of fluorescent-labeled nucleic acid chemistry and the development of oligo-acid probe hybridization technology and the application of real-time technology make the quantitative PCR technology have an adequate basis for the majority of clinical diagnostic laboratories, which will help clinical Doctor's diagnosis and treatment of diseases

Treatment.

4 Conclusion

The development of Real-timeqPCR has enabled researchers to have a simpler and more automated means to study many important fundamental topics. Although there are still some shortcomings in this technology, it has laid a good foundation for the use of real-timeQ-PCR in routine diagnostic testing. oReal-timeQ-PCR will become an indispensable research tool for future molecular biology laboratories.