I. Overview of PCR
1. What is PCR
Polymerase Chain Reaction (PCR), also known as cell-free molecular cloning or in vitro primer-directed enzymatic amplification of specific DNA sequences. Invented by Dr. Mullis of the Genetics Department of American scientist PE(Perkin Elmer Perkin-Elmer), Mullis won the Nobel Prize in Chemistry in 1993 because of the epoch-making significance of PCR technology in theory and application.
2, the history of PCR technology
The principle of PCR is that it is mediated by a pair of primers, and it can carry out rapid enzymatic amplification of specific gene (DNA) fragments in vitro of animals and plants. After N thermal cycles of amplification, the number of specific genes in the amplified products is (1+e) n ( < e < 1, the amplification efficiency = times, which makes it possible to detect specific genes in the amplified products.
the polymerase chain reaction has experienced four generations of products since 1993: the first generation: manual/robotic water bath gene amplification
As shown in the above figure, the temperature of three water baths is kept constant at three temperatures: the high temperature denaturation temperature of PCR (such as 94℃), the low temperature renaturation temperature (such as 58℃) and the appropriate temperature extension temperature (such as 72℃). Then use a basket with PCR specimen test tubes to bathe in water bath boxes with different temperatures in turn by hand, and the constant temperature time of specimens in each water bath box is timed by a stopwatch. In this way, PCR samples can complete the following forms of thermal cycles:
94℃ 3S 58℃ 45S 72℃ 6S
4 cycles
This method is characterized by: the labor intensity of the experimenters is high, and it is easy to make mistakes due to fatigue; The advantages are: the equipment is simple, the investment is low, compared with the automatic gene amplification instrument, it does not need the process of temperature rise and fall, the experiment time is short, and the experiment is closer to the ideal PCR reaction conditions. The fact shows that the experimental effect is still good, but because the specimen tube is exposed to the air for a short time when moving from one water bath box to another, it will also cause temperature interference to the specimen if the moving speed is not fast enough, which will affect the results. Other disadvantages of this method include that it can only be limited to three temperature steps (some PCR reactions need more than three temperature steps), liquid pollution, and it is difficult to burn the water temperature to 94℃ denaturation temperature in low-pressure areas.
In order to improve the automation level of this method, someone designed a manipulator device to replace the above manual sample moving process, and formed a manipulator water-bath gene amplification instrument. This improvement solved the problem of high-intensity labor of experimenters, but it also brought a high fault caused by frequent relative movement of manipulator parts in a long stroke. This type of gene amplifier was sold in Beijing and Shanghai in the mid-199s, and it was widely used. It had made positive contributions to the development of molecular biology in China, and then it was gradually replaced by a more automated amplifier, and now it is rarely used by units. The second generation: automatic control qualitative gene amplification instrument
Compared with the above-mentioned water bath amplification instrument, some people also call this kind of amplification instrument a dry gene amplification instrument, which is the most representative amplification instrument, including the third and fourth generations introduced later, which are all based on the second generation. The third generation: endpoint quantification/semi-quantification
The second generation of qualitative PCR can only judge negative and positive, but can't evaluate the concentration and quantitative analysis of specific nucleic acids. Quantitative PCR can at least achieve the following functions that qualitative PCR can't: Latent virus concentration detection
? Infection degree
? Quantitative change of pathogenic pathogens < P >? Efficacy evaluation of antiviral drugs
? Detection of viral load in recovery period
Since ABI Company of the United States invented the first fluorescent quantitative PCR instrument in 1996, PCR technology and application have developed rapidly from qualitative to quantitative. The advantage of end-point quantitative PCR is less equipment investment. For scientific research units and medical institutions that cannot afford expensive real-time fluorescent quantitative PCR instruments in domestic economic conditions, the existing second-generation conventional qualitative PCR instrument can be used and a special single-hole PCR end-point product fluorescent quantitative detector can be added to start working and play a quantitative role. End-point quantitative PCR technology is an intermediate product in the transition from qualitative to real-time quantitative. However, there are fewer imported products and more domestic instruments, such as TL988 of Xi 'an Tianlong and DA62 of Shanghai Lingguang. The fourth generation: real-time quantitative PCR
real-time quantitative QPCR instrument+real-time fluorescent quantitative reagent+general computer+automatic analysis software, which constitutes a PCR-DNA/RNA real-time fluorescent quantitative detection system.
See the following figure: (omitted)
2. Advantages of real-time fluorescence quantitative PCR:
The equipment consists of a fluorescence quantitative system and a computer, which is used to monitor the fluorescence in the circulation process. A computer connected to a real-time device collects fluorescence data. The data is displayed in the form of charts through the developed real-time analysis software. The raw data is plotted as a graph of fluorescence intensity versus cycle number. After the original data is collected, the analysis can be started. The software of real-time equipment can normalize the collected data to make up for the difference of background fluorescence. After normalization, the threshold level can be set, which is the level of analyzing fluorescence data. The number of cycles that the sample goes through when it reaches the threshold level is called Ct value (the number of cycles at the limit point). The threshold value should be set to maximize the amplification efficiency in exponential period, so that the most accurate and repeatable data can be obtained. If there are also standards labeled with corresponding concentrations at the same time, linear regression analysis will produce a standard curve, which can be used to calculate the concentration of unknown samples.
3. Principle of real-time fluorescence quantitative PCR
The so-called real-time Q-PCR technology refers to the method of adding fluorescent genes to the PCR reaction system, monitoring the whole PCR process in real time by using fluorescence signal accumulation, and finally quantitatively analyzing the unknown template by standard curve. In the development of real-time technology, two important discoveries play a key role: (1) In the early 199s, the discovery of exonuclease activity of Taq DNA polymerase, which can degrade specific fluorescent probes, made it possible to detect PCR products indirectly. (2) After that, the whole reaction process can be monitored in real time in a closed reaction tube by using the fluorescent double-labeled probe. The combination of these two findings and the commercialization of corresponding instruments and reagents led to the application of real-time Q-PCR in research work.
The number of copies of DNA produced in the process of PCR reaction increases exponentially. With the increase of the number of reaction cycles, the final PCR reaction no longer generates templates exponentially, thus entering a platform period. In traditional PCR, gel electrophoresis separation and fluorescence staining are often used to detect the final amplification products of PCR reaction, so it is unreliable to quantify the PCR products by this endpoint method. In real-time Q-PCR, the whole PCR reaction amplification process is monitored in real time and the fluorescence signals related to the amplification are continuously analyzed. With the progress of the reaction time, the changes of the monitored fluorescence signals can be drawn as a curved line. In the early stage of PCR reaction, the level of fluorescence can not be clearly distinguished from the background, and then the fluorescence generation enters exponential period, linear period and final plateau period, so the amount of PCR products can be detected at a certain point in the exponential period of PCR reaction, and the initial content of template can be inferred from this. In order to compare the detected samples conveniently, in the exponential period of the real-time Q-PCR reaction, it is necessary to set a certain threshold value of the fluorescence signal. Generally, this threshold value takes the fluorescence signal of the first 15 cycles of the PCR reaction as the fluorescence background signal, and the default setting of the fluorescence threshold value is 1 times of the standard deviation of the fluorescence signal of 3-15 cycles. If the detected fluorescence signal exceeds the threshold value, it is considered as a real signal, which can be used to define the threshold cycle number (Ct) of the sample. The meaning of Ct value is: the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold value. The research shows that there is a linear relationship between the Ct value of each template and the logarithm of the initial copy number of the template. The more the initial copy number, the smaller the Ct value. The standard curve can be made by using the standard with known initial copy number, so the initial copy number of the sample can be calculated from the standard curve as long as the Ct value of the unknown sample is obtained.
4. Application of real-time fluorescence quantitative PCR in medical treatment
(1) Pathogen detection: At present, pathogens such as Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, human papillomavirus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, influenza virus, Mycobacterium tuberculosis, EB virus and cytomegalovirus can be quantitatively detected by using fluorescence quantitative PCR detection technology. Compared with traditional detection methods, it has the advantages of high sensitivity, less sampling, rapidity and simplicity.
For example, the significance of gene diagnosis of tuberculosis mainly lies in:
a. Distinguishing TB from other mycobacteria;
B. detecting TB drug resistance gene;
C. improve the positive detection rate of TB.
(2) Prenatal diagnosis: Up to now, people can't treat hereditary diseases caused by genetic material changes, so they can only reduce the birth of sick babies through prenatal monitoring to prevent the occurrence of various hereditary diseases. For example, in order to reduce the birth of children with X-linked hereditary diseases, it is a non-invasive method to isolate fetal DNA from the peripheral blood of pregnant women and detect its Y sex-determining region gene by real-time fluorescence quantitative PCR, which is easily accepted by pregnant women.
(3) Evaluation of drug efficacy: The quantitative analysis of hepatitis B virus (HBV) and hepatitis C virus (HCV) shows that the amount of virus is related to the efficacy of some drugs. The high level of HCV expression is insensitive to interferon therapy, while the low titer of HCV is sensitive to interferon therapy. During the treatment of lamivudine, the serum content of HBV-DNA decreased once, and then increased again or exceeded the previous level, which suggested that the virus had mutated. For example, the application and significance of PCR technology in HBV detection: < P > A. Understand the number of hepatitis B virus in the body.
B. whether to copy.
C. whether it is contagious and how contagious it is.
D. Is it necessary to take medicine?
e. whether the abnormal changes of liver function are caused by virus.
F. judge what kind of antiviral drugs are suitable for patients.
g. judge the curative effect of drug treatment.
(4) tumor gene detection: although the mechanism of tumor pathogenesis is not clear, it has been widely accepted that the mutation of related genes is the fundamental cause of carcinogenic transformation. The increase and mutation of oncogene expression can appear in the early stage of many tumors. Real-time fluorescence quantitative PCR can not only effectively detect gene mutation, but also accurately detect the expression of oncogene. At present, this method has been used to detect the expression of telomerase hTERT gene, chronic myeloid leukemia WT1 gene, tumor ER gene, prostate cancer PSM gene, tumor-related virus gene and so on. With the discovery of new genes related to tumor, fluorescence quantitative PCR technology will play a greater role in tumor research.
(5) Application in prenatal and postnatal care:
In recent years, the economy has developed rapidly, people's living standards have improved year by year, and more and more attention has been paid to the health of themselves and their families, especially the next generation. In addition, due to the deepening of family planning work in China, there are more only children, and the physical quality of children has become the focus of attention of elders. Therefore, how to improve the quality of newborns and human genetic quality, that is, prenatal and postnatal care, has become very important. ① Avoid the birth of individuals with serious hereditary diseases and congenital diseases. ② Promote the reproduction of individuals with excellent physical strength and intelligence. Among them, avoiding the birth of individuals with serious genetic diseases and congenital diseases is the most basic content of prenatal and postnatal care. From the point of view of clinical eugenics, the specific work includes genetic examination of mother and fetus during pregnancy, trying to exclude the birth of common hereditary diseases, and checking whether mother has some infectious diseases such as Toxoplasma gondii, rubella virus and chlamydia that are easy to cause fetal malformation during pregnancy. In the past, chromosome analysis was mainly used to detect hereditary diseases, but most hereditary diseases in clinic were genetic diseases rather than chromosomal diseases, which could not be detected by chromosome analysis. If PCR gene amplification combined with single strand conformation polymorphism analysis (sscp), restriction fragment length polymorphism analysis (RFCP), allele-specific oligonucleotide acid (Aso) dot hybridization or differential PCR is used, the mutation of a single gene can be easily detected, and the accuracy can reach over 95%, so this problem has been solved well. Common pathogens of infectious diseases that easily cause fetal malformation are herpes simplex virus (HSVⅡ), rubella virus (RV), human cytomegalovirus (HCMV), TOX and Chlamydia trachomatis (CT). In the past, it was difficult to diagnose these pathogen infections, and the diagnosis was mainly made by culture method. However, culture method was time-consuming and expensive, and was affected by sampling, sample preservation and medication, so it could not be popularized in clinic. Because of its high sensitivity and specificity, PCR gene amplification method is very suitable for the diagnosis and curative effect tracking of these diseases, and it is the most recommended test method.
1. Application of PCR gene amplification method in prenatal diagnosis of hereditary diseases. Hereditary diseases are diseases caused by a certain function, defect or abnormality of the organism caused by genetic changes, and the fundamental change lies in genetic materials. Its types include monogenic genetic diseases, chromosomal genetic diseases and polygenic genetic diseases. The diagnosis of genetic diseases has its particularity except asking about medical history, general physical diagnosis, general laboratory examination and understanding symptoms and signs. In the past, pedigree analysis, chromosome and sex staining were often used as the main basis for the diagnosis of genetic diseases, supplemented by related enzymatic analysis to make a diagnosis. With the rapid development of molecular biology and its wide application in the diagnosis of hereditary diseases, gene diagnosis technology was born, which greatly promoted the clinical diagnosis of hereditary diseases. Polymerase chain reaction (PCR) is one of the main techniques in gene diagnosis. This rapid and sensitive gene amplification technology in vitro can detect most known gene mutations, gene deletions, chromosome dislocations, etc., and PCR technology is increasingly becoming one of the most effective and reliable methods for the diagnosis of genetic diseases. Here are a few examples of universal significance in China.
(1) Detection of thalassemia by PCR; Thalassemia is a hereditary chronic hemolytic anemia, which is the most common and the most common single-gene genetic disease in the world. In Guangdong, Guangxi, Guizhou, Sichuan and other places, the incidence rate is high, up to 15% in Guangxi and other places. Thalassemia is due to the imbalance of globin caused by gene mutation, which makes the structure normal.