Early PCR is mainly used for qualitative analysis, and it is called terminal PCR according to the presence or absence of target sequence, which is widely used in gene identification, pathogenic nucleic acid detection and other fields.
In 1990, Simmonds et al. roughly determined the copy numbers of HCV, HIV and other pathogens through gradient dilution of terminal products, which may be the earliest quantitative PCR study. However, it needs to be clarified that the quantitative PCR mentioned here does not refer to fluorescent quantitative PCR, and fluorescent substances were not used to monitor PCR products at that time. Until 1992, R Higuchi of Roche published a paper in Nature, introducing the method of dynamically monitoring PCR products by using ethidium bromide (EB), which may be the earliest fluorescence quantitative PCR technology. 1996, ABi company announced qPCR technology based on Taqman probe. In 1997, Wittwer et al. compared the characteristics of qPCR based on (i) double-stranded specific dye SYBR Green I, (ii)5'- nuclease and double-labeled probe, and (iii) molecular beacon Cy5. These studies laid the foundation for the wide application of qPCR later.
Generally people are used to divide QCPR into relative quantization and absolute quantization, but in essence, QCPR can only be used for relative quantization, and the realization of absolute quantization often needs the help of "external force". PCR amplification is exponential amplification. In an ideal state, the product concentration has the following relationship with the initial concentration: N T = N 0 ×2 n (N 0 represents the initial concentration, N T represents the final concentration, and N represents the cycle number), and both sides of the formula can be obtained by logarithm: log N T = logN 0+n×log2(log represents the logarithm based on the natural constant E). If we fix the judgment signal of qqPCR endpoint to a unified value (that is, the fluorescence threshold in QCPR), then the logarithm of cycle number and initial concentration becomes linear, which is the basic principle of QCPR relative quantification. However, there are two factors: (1) The human eye can't accurately judge the PCR endpoint signal, so a special equipment-fluorescent PCR instrument appears; (2) The amplification efficiency of different target genes is different, so it is impossible to compare them directly, so Ct method was born.
Real absolute quantitative PCR is called digital PCR(dPCR, 1999 Kinzler and others first put forward the concept of digital PCR), which is an absolute quantitative method to calculate the copy number through Poisson distribution on the basis of terminal PCR and extreme dilution. In the research of Simmonds et al., they diluted the DNA molecule to a single copy, and then calculated the molecular number of the target gene according to the terminal signal of PCR and Poisson distribution law. However, they didn't further develop this technology, which was applied with the characteristics of molecular counting for a long time. On the one hand, dPCR has been suppressed by QCPR for a long time, and on the other hand, it is limited by testing instruments. It was not until 2006 that DPCR gradually showed the scene of technical recovery.
In 1993, Zachar et al. introduced the mathematical principle of relative quantification of target genes by PCR in nucleic acid research. In 200 1, Livak KJ et al. introduced the derivation process, limitations and applications of 2-Ct method.
Of course, these principles are simple and easy to understand even without reading the paper. Because of the exponential amplification of PCR, when we fix the criteria for judging the end point, the samples with high initial template amount arrive first, and the samples with low initial template amount consume more cycles, each cycle means that the initial concentration is 2 times different, that is, N 1/N2 = 2 -(Ct 1-Ct2). When detecting different samples, Ct may be affected by the difference of sample size, so the correction of internal reference gene is introduced. Internal reference genes, also known as housekeeping genes or housekeeping genes, are generally considered to maintain constant expression in different time and space tissues of organisms. So what are the internal reference genes of the two samples? Ct represents the difference of sample size, target gene? CT- internal reference gene? Ct is the true expression difference of the target gene, which is a 2-Ct method.
However, there are several problems to be noted: (1)PCR is not all exponential growth period, and comparison must be carried out in logarithmic growth period; (2) In general, the default amplification efficiency in logarithmic growth period is 100%, which is not rigorous, especially for some templates which are difficult to amplify, the efficiency may be quite different from 100%, so the expression of a gene (such as gene A) is analyzed vertically. Still using 2-Ct may not be accurate. (3) The amplification efficiency of different target genes is different, so when comparing different target genes horizontally, it may cause a big error.
In view of this, when designing primers, we should try to keep the target length, GC% and Tm close to each other, so as to ensure similar amplification efficiency. PrimerBank and qPrimerDB are excellent QCPR primer retrieval websites at home and abroad respectively, which contain a large number of QCPR primer data of species and have certain reference value. In addition, Pfaffl et al. (200 1) and Rao(20 13) modified the 2-Ct method, and the method adopted was mainly to correct the amplification efficiency by diluting the same template gradient, which has certain reference significance.
There are two methods for absolute quantification of qPCR: (1) First, get a reference gene with a known copy number, then get the ratio of the target gene to this reference, and then get the accurate number of test samples according to the known value. From this perspective, absolute quantization is relative quantization with the help of the external force of "known copy number". In 1990, gilliland et al. described this principle. (2) According to the method reported in (2) Symonds, the DNA template was diluted to the limit until the PCR system only contained one template molecule. At this time, the copy number of the target gene in the sample can be obtained by multiplying it by the dilution multiple. This method is the technical prototype of dPCR, and it is very difficult in practical operation. One is that many dilution gradients are needed, and the other is that it is often difficult to amplify the common 10-20uL system with only one template molecule.
The most extensive application of absolute quantification is molecular counting, such as the accurate determination of RNA molecular number and the identification of gene copy number in DNA genome. Southern hybridization is the most widely used method to identify the copy number of foreign genes. However, with the continuous development of QCPR technology, there are more and more reports on copy number identification based on QCPR absolute quantization. A large number of studies show that the results obtained by QCPR method are basically the same or even more accurate. Song et al. (2002) used qRT-PCR to estimate the transgenic copy number in transgenic maize calli and plants, and this study also used Southern hybridization to re-measure the "accurate" transgenic copy number in maize calli and plants. Therefore, the results of qRT-PCR measurement are highly correlated with the "accurate" results, so they think qRT-PCR can be used as an effective means to evaluate the copy number of transgenic corn.
The key of copy number identification is to obtain the standard with known copy number. Plasmids are easy to extract and purify, so they are often used to construct absolute quantitative standards. Purify the recombinant plasmid carrying the target gene to a very high purity, accurately determine its nucleic acid concentration, and calculate the standard copy number according to the formula: n = 6.02× 1023 (copy/mole )× mdna (gram) /(DNA length (BP )× 650 (gram/mole /BP), where n represents the number of molecules. Draw the standard curve of logN and ct with this standard, and then calculate the exact number of target genes according to the Ct value of target genes. At the same time, we need to select a known reference gene from the genome, draw a standard curve, count the molecules in the same way, and then determine the molecular numbers of the target gene and the reference gene in the unified sample, and bring them into the above formula to get the actual copy number of the target gene. Generally, genes with low copy number and high conservation should be selected as reference genes.
There are many forms of copy number identification, and double standard curve is not necessary. If it can be confirmed that the amplification efficiency of the target gene and the reference gene is close to 100%, the copy number of the target gene can also be determined by 2-Ct method. Lin Weishi et al. (20 13) used this method to obtain the copy number identification result consistent with Southern hybridization.
There are many methods for genotyping. Landegren et al. (1998) summarized a variety of genotyping techniques, among which qPCR and sequencing are the most widely used methods. Sequencing method is the most accurate and can find new genotypes. It is the gold standard for genotyping or SNP detection, but it is slow and complicated to operate. QPCR detection is simple and extremely fast, and it is widely used at present.
The basic principle of qPCR genotyping is that 3'- terminal mismatched primers cannot amplify the target gene normally. In 1989, Wu et al. and Newton et al. successively reported the detection of alleles by ASPCR method. This method is easy to understand. Assuming that the SNP site is known as A/T, if the terminal signal produced by PCR product of 3'-A primer can be judged as genotype, the terminal signal produced by 3'-T primer is T genotype, and the signals produced by both primers are heterozygous. In 1995, Livak et al. reported the method of detecting SNP with probes with different fluorescent labels. In this method, two probes with different fluorescent labels were designed for two genotypes, and pure products and genotype controls were set up. Amplified by PCR, if the fluorescence signal is close to the A reference, it represents the A genotype, and if it is between A and B, it is a heterozygote (as shown in the following figure).
In 2003, Papp et al reported a SNP typing method based on high resolution dissolution curve, which was also based on 3'- terminal mismatched primers. Genotype A designed a primer with normal length, while genotype B added a high GC sequence of 10- 15bp at the 5'- end of the primer. After QCPR amplification, the Tm of different genotype products will change, which depends on the high resolution of QCPR instrument.
After 1995, the number of qPCR related research papers increased exponentially and became one of the hottest fields in molecular biology. In recent years, with the rise of molecular diagnosis industry, qPCR has played an increasingly important role in the medical field. With the rapid development of qPCR, there are also some problems, such as inconsistent judgment standards, no unified standard for detection accuracy, and serious false positives in RNA detection. In 2009, many scientific research institutes and medical institutions jointly issued the MIQE guide of QCPR, which standardized the commonly used terms of QCPR, such as Ct should be called Cq, RT-qqpcr should be written as RT-QCPR, etc. And standardize the sensitivity, specificity and accuracy of the analysis. In addition, the guide also provides detailed specifications for sample processing, nucleic acid extraction, reverse transcription, QCPR and even data analysis. The guide consists of 9 parts and 85 parameters to ensure the practicality, accuracy, correctness and repeatability of qPCR experiment. Although the guide has existed for several years, following these specifications can make your research easier to repeat and help reviewers and editors evaluate your manuscript quickly.
Note: the contents and timetable of the guide can be obtained here: competitive PCR for efficient analysis of multiple samples. Nucleic acid research1993; 2 1(8):20 17‐20 18.doi: 10. 1093/NAR/2 1.8 . 20 17
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