Biology is divided into two eras: the pre-PCR era and the post-PCR era, which is what the New York Times said about Mr. Mullis's invention of PCR technology.In 1983, Kary B. Mullis came up with the idea of PCR technology, and in 1985, they published a paper about it in Science. The paper was led by Randall K. Saiki, a colleague of Mullis. In 1988, Saiki et al. isolated and purified Taq DNA polymerase and applied it to PCR reaction, which made PCR simpler, easier and more stable, and then PCR technology ushered in a period of vigorous development. PCR has roughly gone through the following three phases according to the accuracy of its analysis: (I) Endpoint PCR: qualitative analysis (II) Quantitative PCR: relative quantification (III) Digital PCR: absolute quantification
Early PCR is mainly used for qualitative analysis, based on the presence or absence of the reaction endpoint product to detect the presence or absence of target sequences, this PCR can be referred to as the endpoint PCR, which is widely used in the field of gene identification, pathogenic nucleic acid detection and other fields.
In 1990, Simmonds et al. conducted a rough copy number identification of HCV, HIV and other pathogens by gradient dilution of the endpoint product, which is probably the earliest quantitative PCR study. It should be clarified, however, that quantitative PCR in this context does not yet refer to fluorescent quantitative PCR, and the use of fluorescent substances for PCR product monitoring was not yet available. It was not until 1992 that R Higuchi et al. of Roche published a paper in Nature describing the use of ethidium bromide (EB) for dynamic monitoring of PCR products, which was probably the earliest fluorescent quantitative PCR technique. in 1996, ABi published a qPCR technique based on Taqman probes. in 1997, Wittwer et al. compared (i) a double-stranded specific dye-based qPCR technique with a double-stranded specific dye-based qPCR technique. ) the characteristics of qPCR based on the double-stranded specific dye SYBR Green I (ii) 5'-nuclease-based and double-labeled probes (iii) Cy5-based molecular beacons. These studies laid the foundation for the later widespread use of qPCR.
Generally, qPCR is categorized into relative and absolute quantification, but essentially, qPCR can only be used for relative quantification, and absolute quantification is often achieved with the help of "external forces". PCR amplification is a kind of exponential amplification, ideally, the product concentration and the starting concentration there is the following relationship: N T = N 0 × 2 n (N 0 on behalf of the starting concentration, N T on behalf of the endpoint concentration, n on behalf of the number of cycles), on both sides of the formula to take the logarithm of the formula can be obtained: log N T = logN 0 + n × log2 (log on behalf of the logarithm of the bottom of the natural constant e), if we take the endpoint of PCR judgment signal fixed into a uniform value, then we will be able to achieve absolute quantification. judgment signal fixed to a uniform value (i.e., the fluorescence threshold in qPCR), then the number of cycles becomes linear with the logarithm of the starting concentration, which is the basic principle of relative quantification by qPCR. However, there are two factors: (1) the human eye cannot accurately judge the PCR endpoint signal, so special equipment-fluorescence PCR instrument appeared; (2) different target genes have different amplification efficiencies, so it is not possible to compare them directly, so it gave birth to the ? Ct method.
True absolute quantitative PCR is called digital PCR (dPCR, the concept of digital PCR was first introduced by Kinzler et al. in 1999), which is an absolute quantitative method to derive copy number by Poisson distribution calculation based on endpoint PCR and limiting dilution. In Simmonds et al.'s study, they calculated the number of molecules of target genes by diluting DNA molecules to a single copy and then calculated the number of molecules of target genes based on the end-point signals of PCR and the law of Poisson distribution, although they did not develop the technique further, and for a long time it was applied with the characteristics of molecule counting. dPCR was on the one hand suppressed by the long time suppression of qPCR, and on the other hand by the dPCR was long suppressed by qPCR on the one hand, and by the limitations of the detection instruments on the other, and it was not until after 2006 that the technology showed a gradual resurgence.
In 1993, Zachar et al. introduced the mathematical principles of relative quantification of target genes using PCR in Nucleic Acids Research; in 2001, Livak KJ et al. introduced the 2 - ? Ct method derivation process, limitations and applications.
Of course these principles are simple and easy to understand even without reading the papers. Because of the exponential amplification of PCR, when we fix the criterion for judging the endpoint, samples with high starting template amount are the first to arrive, samples with low starting template amount consume more number of cycles, and each cycle difference represents a 2-fold difference in starting concentration, i.e., N1/N2 = 2 - (Ct1-Ct2) . When testing different samples, ? Ct may be affected by differences in sample size, so a correction for the internal reference gene is introduced. The internal reference genes, also called housekeeping or caretaker genes, are generally considered that they maintain constant expression in different spatiotemporal tissues of the organisms, then the two samples of internal reference genes ? Ct then represents the difference in sample size, the target gene's ? Ct - the ? Ct is the real expression difference of the target gene, which is the 2 - ? Ct method.
However, there are several issues to note: (1) PCR is not always exponential growth period, the comparison must be made in the logarithmic amplification period (2) the general default amplification efficiency of the logarithmic growth period is 100%, which is not rigorous, especially some of the amplification of difficult templates, the efficiency of the efficiency of 100% may be very different from the longitudinal analysis of the expression of a particular gene (such as the expression of gene A in different stages of production / different tissues), but still the expression of the target gene is not the same as that of the target gene. tissue expression), the use of 2 - ? Ct may not be very accurate (3) the amplification efficiency of different target genes is different, so when comparing different target genes horizontally, it may result in a large error.
In view of this, we should keep the length, GC%, and Tm of the targets as close as possible when designing primers to ensure similar amplification efficiency. PrimerBank and qPrimerDB are respectively the better qPCR primer search websites in foreign and domestic countries, which contain qPCR primer data of a large number of species with certain reference value. In addition, Pfaffl et al. (2001), Rao (2013) and others have made some corrections to the 2 - ? Ct method with some corrections, the method used is mainly amplification efficiency correction by gradient dilution of the same template, which has some reference significance.
There are two methods for absolute quantification of qPCR: (1) to obtain a reference gene with a known copy number, and then to obtain the ratio of the target gene to the reference, and then to obtain the exact number of samples to be tested according to the known value, and from this point of view, absolute quantification is the relative quantification with the help of the external force of "known copy number". In 1990, Gilliland et al. described this principle. (2) In the method reported by Simmonds et al., the DNA template is diluted to the limit until the PCR system contains only one template molecule, at which time the copy number of the target gene in the sample can be obtained by multiplying the number of dilutions. This method is the prototype of dPCR technology, which is very difficult in practice, firstly, it requires a lot of dilution gradients, and secondly, it is often difficult to amplify successfully with only one template molecule in an ordinary 10-20uL system.
The most widespread application of absolute quantification is molecular counting, such as the precise determination of RNA molecules, and the identification of gene copies on the DNA genome, etc. Southern hybridization is the most widely used method for exogenous gene copy number identification, but with the continuous development of qPCR technology, the reports of copy number identification based on absolute quantification by qPCR have been increasing gradually, and a large number of studies have shown that the qPCR method is comparable to the Southern hybridization method. Song et al. (2002) used qRT-PCR to estimate the transgene copy number in transgenic maize healing tissues and plants, and also used Southern hybridization to re-measure the "exact" transgene copy number in maize healing tissues and plants. The study also used Southern hybridization to re-measure the "exact" transgene copy number in maize healing tissues and plants, and the results of the qRT-PCR measurements showed a high correlation with the "exact" results, and therefore, they concluded that qRT-PCR can be an effective means of evaluating the copy number of transgenic maize.
The key to copy number identification is to obtain a standard with a known copy number. Plasmids are easy to extract and purify, so they are often used to construct standards for absolute quantification. The recombinant plasmid carrying the target gene is purified to a very high purity, and its nucleic acid concentration is accurately determined. The copy number of the standard can be calculated according to the formula: N = 6.02 × 10 23 (copy/mol) × M DNA (g) / (DNA length (bp) × 650 (g/mol/bp)), where N represents the number of molecules and M DNA represents the weight of the plasmid. The standard curve of logN versus Ct is plotted with this standard, and then the exact number of target genes can be inverted according to the Ct value of the target genes. At the same time, we have to choose a reference gene from the genome whose gene copy is known, draw the standard curve and count the molecules in the same way, and then determine the number of molecules in the unified sample between the target gene and the reference gene, and then bring them into the above formula to get the actual number of copies of the target gene. Generally, the genes with low copy number and high conservation within the species should be selected as reference genes.
There are many forms of copy number identification, and a double standard curve is not necessary. If you can confirm that the amplification efficiency of both the target gene and the reference gene is close to 100%, you can also use the 2 - ? Ct method can also be used to measure the copy number of the target gene if it can be confirmed that the amplification efficiency of both the target gene and the reference gene is close to 100%. Lin Weishi et al. (2013) obtained copy number identification results consistent with Southern hybridization by this method.
There are many methods for genotyping, and Landegren et al. (1998) reviewed a variety of techniques used for genotyping, of which the most widely used are qPCR and sequencing. Sequencing is the most accurate and can detect new genotypes, and is the gold standard for genotyping or SNP detection, but it is slow and cumbersome. qPCR is easy to perform and very fast, and is now very widely used.
The basic principle of genotyping by qPCR is that 3'-end mismatched primers cannot amplify the target gene properly. 1989, Wu et al. and Newton et al. successively reported the ASPCR method and the method for detecting alleles, which is easy to understand, assuming that the known SNP locus is A/T, and if the 3'-A primer PPCR is used, it is not possible to detect the target gene. '-A primer PCR product produces an endpoint signal can be judged as the A genotype, 3'-T primer produces an endpoint signal for the T genotype, both primers produce signals that is heterozygous.In 1995, Livak et al. reported a method of SNP detection using probes with different fluorescent markers, in this method, respectively, for the two kinds of In this method, two probes with different fluorescent markers were designed for two genotypes, and pure and genotypic controls were set up. With PCR amplification, if the fluorescent signal is close to the A reference, it represents the A genotype, and close to the B reference represents the B genotype, and if it is located in the middle of A and B, it is the heterozygous genotype (as shown in the figure below).
In 2003, Papp et al. reported a SNP typing method based on high-resolution dissolution curves, which was also based on 3'-end mismatched primers, with the A genotype designing normal-length primers, and the B genotype adding 10-15bp high-GC sequences at the 5' end of the primers, and after After PCR amplification, the Tm of the products of different genotypes will change, and relying on the high-resolution solubilization curve of the qPCR instrument, the genotypes can be quickly distinguished.
After 1995, the number of qPCR-related research papers grew exponentially, becoming one of the hottest areas of molecular biology. In recent years, with the rise of the molecular diagnostic industry, qPCR has played an increasingly important role in the medical field. qPCR's rapid development has also generated a number of problems, such as inconsistent judgment criteria, no uniform standards for detection accuracy, and more serious false positives in RNA detection, etc. In 2009, a number of scientific research institutes and medical units collaborated to release the MIQE Guidelines for qPCR. MIQE Guidelines, which standardize the common terminology of qPCR, such as Ct should be called Cq, RT-PCR should be written as RT-qPCR, etc., and standardize the sensitivity, specificity, precision of the analysis, etc., in addition to the guidelines for the sample processing, nucleic acid extraction, reverse transcription, qPCR, and even the data analysis are all exhaustive specifications. The guideline consists of 9 parts***85 parameters to ensure the practicality, accuracy, correctness and reproducibility of qPCR experiments. Although the guidelines are a bit old, adherence to these specifications will make your studies more reproducible and will help reviewers and editors to quickly evaluate your manuscript.
Note: The content and schedule of the guidelines are available here: petitive PCR for efficient analysis of multiple samples. Nucleic Acids Res. 1993;21(8):2017-2018. doi. 10.1093/nar/21.8.2017
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