Digital PCR is Digital PCR (dPCR), which is an absolute quantification technology of nucleic acid molecules. Compared with qPCR, digital PCR allows you to directly count the number of DNA molecules, which is an absolute quantification of the starting sample.
Digital PCR is a quantitative analysis technique that has developed rapidly in recent years. The result of this technique does not depend on the cycle threshold (Ct) of the amplification curve, is not affected by the amplification efficiency, has good accuracy and reproducibility, and can achieve absolute quantitative analysis. Digital PCR has shown great technical advantages and application prospects in research fields such as nucleic acid detection and identification. This push mainly introduces the history and development of dPCR, the basic principles of dPCR, and the commercial dPCR platform and its characteristics.
Introduction
In quantitative PCR, we often struggle with a question, is it relative quantification or absolute quantification? Now, you don't need to worry about it, because digital PCR (digital PCR) is here. Although these two techniques are somewhat similar and both estimate the amount of nucleic acid in the starting sample, there is an important difference between them. Quantitative PCR relies on a standard curve or reference gene to determine the amount of nucleic acid, while digital PCR allows you to directly count the number of DNA molecules, which is an absolute quantification of the starting sample. Therefore, it is especially suitable for application fields that cannot be distinguished by Ct value: copy number variation, mutation detection, relative gene expression research (such as allele imbalanced expression), second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression Analysis etc.
History and development of dPCR
Digital PCR, or Digital PCR (dPCR), is an absolute quantification technology of nucleic acid molecules. Compared with qPCR, digital PCR can directly read the number of DNA molecules, which is an absolute quantification of the nucleic acid molecules of the starting sample. dPCR originated from the method first published by Cetus Corporation in 1988, when researchers demonstrated that a single β-globin molecule can be detected and amplified by PCR. This is done by dividing the sample, so some reactions contain the molecule while others do not. In 1990, Peter Simmonds and AJ Brown first used this concept to quantify molecules. Alex Morley and Pamela Sykes formally established this method as a quantitative technique for nucleic acids in 1992.
In 1999, Bert Vogelstein coined the term "digital PCR". The article was formally reported in PNAS, Proceedings of the National Academy of Sciences, and showed that the technology can be used to find rare cancer mutations. The author is American cancer researcher and Howard Hughes Medical Institute researcher Bert Vogelstein (Bert Vogelstein).
By dividing a sample into tens to tens of thousands of copies and assigning them to different reaction units, each unit contains at least one copy of the target molecule (DNA template). The target molecule is amplified by PCR in each reaction unit. After the increase, the fluorescence signal of each reaction unit is statistically analyzed.
The article uses the detection of KRAS gene mutations in the stool of patients with colon cancer, and highlights the ability and potential of dPCR quantitative detection (Because PCR is an exponential process, qPCR can only observe twice the amplification difference, but the article highlights The ability of dPCR to distinguish such small differences). However, the article only distributes the samples to 384-well plates, detects mutant and normal genes with different fluorescence, and calculates the mutation rate by calculating the ratio of mutant and normal genes. Although it is necessary to distribute samples into more micropores or droplets in order to be able to improve detection performance, the basic idea is inseparable from the dPCR concept established by Bert Vogelstein.
In 2003, Kinzler and Vogelstein continued to perfect dPCR and created an improved method, which they called BEAMing technology, the acronym for "beads, emulsions, amplification, and magnetism." The BEAMing technology uses emulsions to separate amplification reactions in a single tube. This change can scale the PCR to thousands of reactions in a single run.
Since then, the basic methods for the two forms of dPCR (chip type and droplet type) have been established. In terms of commercialization of dPCR, manufacturers such as Bio-Rad, LIFE Technologies, and RainDance have successively introduced more mature digital PCR products.
QuantaLife's microdrop digital PCR technology. The product also won the 2011 Frost & Sullivan North American New Product Innovation Award. In October 2011, Bio-Rad acquired QuantaLife and ddPCR technologies, and successively launched QX100 and QX200 droplet digital PCR systems.
Basic principles of dPCR
At present, dPCR mainly has two forms, chip type and droplet type, but the basic principle is to disperse a large amount of diluted nucleic acid solution into the microreactors or droplets of the chip, and the number of nucleic acid templates in each reactor is less than or Equal to 1. In this way, after the PCR cycle, a reactor with a nucleic acid molecule template will give a fluorescent signal, and a reactor without a template will have no fluorescent signal. According to the relative ratio and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.
Several different methods can be used to dispense samples, including microplates, capillaries, oil emulsions, and miniaturized chamber arrays with nucleic acid binding surfaces. The distribution of samples allows people to estimate the number of different molecules by assuming that the molecular population follows the Poisson distribution. According to the principle of Poisson distribution, the copy number of the target molecule in the reaction system can be obtained by the formula A=-ln[(NX)/N] *N calculation, thus solving the possibility that multiple target molecules exist in a single droplet.
At the same time, it can be seen from the formula that as the number of positive reaction systems (X) increases, the copy number of the target molecule in the system will have a larger gap with respect to X. As X continues to increase, the digital PCR results will not The degree of certainty has also increased. Generally speaking, the number of digital PCR positive systems shall not exceed 80% of the total number of systems. On the other hand, the increase of N will make the entire digital PCR system have a larger linear range, and can improve the sensitivity, stability and repeatability of the reaction. Therefore, the current dPCR needs to increase the number of chambers and droplets allocated while the cost is controllable.
Commercial dPCR platform and its characteristics
Up to now, there are mainly two common dPCRs on the market: droplet dPCR (dPCR, ddPCR) and chip dPCR (chip dPCR, cdPCR) technology. Due to the high cost of chip-based dPCR manufacturing chips, the droplet-based dPCR is now increasingly recognized by enterprises.
cdPCR (chip type) is mainly represented by Fluidigm's BioMark HD system and Life Technology's QuantStudio 3D system. The Bio Mark system uses a micro-pump valve chip and polydimethylsiloxane as the chip material. It mainly relies on the opening and closing of the microfluidic channel and the valve to perform the original system segmentation. The PCR reaction is carried out in the reaction chamber of the chip, and then similar The method of gene chip scans the fluorescent signal of each through hole to calculate the content of the target sequence. The QuantStudio 3D system adopts an array micro-pool type chip, and the reaction liquid enters each micro-reaction cell directly from the injection hole. Chip-type dPCR generates uniform droplets in volume, has high stability, and has little influence between systems, but the technical operation is complicated, the throughput is limited, and the experimental cost is high.
ddPCR (microdrop type) mainly includes the QX200 system and the Rain Drop system developed by Bio-rad. The QX200 can divide the system into 20,000 droplets, and the Rain Drop can be divided into 1 million to 10 million droplets. The principle of ddPCR is to process a PCR reaction system to be analyzed into droplets, and use a droplet generator to make nearly 20,000 water-in-oil droplets to segment the original system. The nucleic acid molecules in the sample are randomly allocated to In a large number of independent droplets, each droplet contains one or no nucleic acid molecule to be tested. After the amplification reaction of the droplet system, the fluorescence signal of each droplet is analyzed, and the presence or absence is judged. The judgment result is determined according to the principle of Poisson distribution by reading the number of positive droplets of the target and internal reference nucleic acid and The ratio thus obtains the copy number and concentration of the target molecule. Compared with chip-based dPCR, the operation is simple, high-throughput detection can be realized, and the stability of droplet detection is guaranteed to a certain extent.
Richard Kurtz, sales manager of Bio-Rad's gene expression department, said that the company's unique advantage of ddPCR is the ability to produce very uniform, repetitive 1 nanoliter droplets. The advantage of this is that each sample forms 20,000 droplets, while other systems can only be divided into 760-3,000 parts. The more points you get, the more accurate the analysis.
Advantages of digital PCR
Digital PCR is an absolute quantification technology of nucleic acid molecules. There are currently three methods for the quantification of nucleic acid molecules: (1) Photometric method is based on the absorbance of nucleic acid molecules; (2) Real-time PCR (Real Time PCR) is based on the Ct value, which refers to the cycle corresponding to the fluorescence value that can be detected Number; (3) Digital PCR is the latest quantitative technology, based on the single-molecule PCR method for nucleic acid quantification, which is an absolute quantitative method. Mainly adopt the microfluidic or dropletization method in the current popular research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactor or droplet of the chip, and the number of nucleic acid templates in each reactor is less than or equal to 1 A. In this way, after the PCR cycle, a reactor with a nucleic acid molecule template will give a fluorescent signal, and a reactor without a template will have no fluorescent signal. According to the relative ratio and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.
Compared with conventional PCR methods, dPCR has very good advantages.
Absolute quantification
Both conventional PCR and real-time fluorescent PCR quantitative detection require a known copy number of standard DNA to develop a standard curve. Because the sample determination will not be completely consistent under various conditions, it will cause differences in PCR amplification efficiency, which will affect the accuracy of quantitative results. . However, ddPCR is not affected by the standard curve and amplification kinetics and can be used for absolute quantification.
Low sample demand
It has obvious advantages when detecting precious samples and sample nucleic acid degradation.
High sensitivity
ddPCR essentially divides a traditional PCR reaction into tens of thousands of independent PCR reactions. In these reactions, small differences in target fragments, single copies or even low-concentration mixed samples can be accurately detected, and non-uniformity can be avoided. The formation of homologous double strands.
High tolerance
Since the target sequence is distributed into multiple droplets, the influence between the systems and the interference of the background sequence and inhibitors on the reaction are significantly reduced, and the amplification matrix effect is greatly reduced.
The specific comparison of each PCR is shown in the following table:
At the same time, dPCR also has some shortcomings, such as high cost of digital PCR system, limited throughput, and cumbersome operation. Due to the high cost of chip processing and the high system complexity of chip-based dPCR, the commercialization advantage is not particularly obvious. In particular, most of the current molecular diagnostic qPCR can also meet the demand well, and the increased revenue-cost ratio of dPCR is not high. The cost of the droplet type is lower than that of the chip type. However, the typical droplet type dPCR droplet formation and PCR amplification and detection are completed in different instruments, which increase a lot of operations and increase the complexity of the system. Look at the point of view that fully automated fluorescent quantitative PCR is doing better.
summary
Although dPCR is called the third-generation PCR technology, the wide application of dPCR technology gradually shows some shortcomings. The throughput is not high, the operation is complicated, and the cost is greatly increased. It seems that dPCR has not found enough irreplaceable advantages over qPCR. On the other hand, dPCR adds a lot of technical difficulties, how to make low-cost chips, how to integrate droplet generation and PCR amplification, how to perform sensitive signal detection and analysis, how to improve the degree of automation, and realize Sample-In Result-Out. Compared with Digital ELISA, which can increase the sensitivity of immunoassays to the fg/mL level and realize the detection of highly sensitive proteins, although dPCR has been developed earlier, it is difficult to obtain Killer Application. Perhaps this has also led to the so-called third-generation PCR in many people. It seems that it is not so "next generation".