What is the difference between ordinary PCR technology and digital PCR technology?
Since the advent of PCR technology, it has played a huge role in the field of molecular diagnosis of genetic diseases, pathogens, oncogenes, and forensic identification. According to the evolution of PCR technology, people habitually divide PCR technology into three generations: ordinary PCR technology, real-time fluorescent quantitative PCR technology (qPCR) and digital PCR (dPCR) technology. I have also introduced a lot of knowledge about qPCR in the past. In this issue, I will share the other two PCR technologies-ordinary PCR technology and dPCR technology, in order to let everyone have a better understanding of these technologies.
Ordinary PCR
1. What is PCR
PCR (Polymerase Chain Reaction, polymerase chain reaction) is an in vitro nucleic acid amplification system. Its principle is similar to the natural replication process of DNA molecules. It combines the DN fragment to be amplified and two complementary oligomers on both sides of it. Nucleotide primers, after several cycles of denaturation, annealing and extension, amplify the DNA by 2n times. This technology has become an essential tool in molecular biology that helps DNA cloning and gene analysis.
2. Basic principles of PCR
The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity relies on oligonucleotide primers complementary to both ends of the target sequence. This technology relies on the enzymatic synthesis reaction of DNA polymerase in the presence of template DNA, primers, and 4 kinds of deoxynucleotides, and passes the DNA fragment to be amplified with the primers of the oligonucleotide strands complementary to both sides of it. The multiple cycles of the three-step reaction of "high temperature denaturation-low temperature annealing-primer extension" increase the number of DNA fragments exponentially, so that we can obtain a large number of specific gene fragments in a short time. The semi-reserved replication of DNA is an important way for biological evolution and passage.
3. PCR reaction elements
①DNA template: it can be double-stranded, single-stranded, chromosome extracted DNA, cloned plasmid DNA; ②Primers: a pair of oligo DNA, which are complementary to the 3'-end sequences on both sides of the template, so that the DNA sequence can be amplified; ③DNA polymerase (TaqDNA polymerase): catalyzes DNA synthesis; ④Buffer: provides the pH, ionic strength and other environments required for DNA synthesis reactions; ⑤4 types of single nucleotides: dNTPs, which provide raw materials for DNA synthesis.
Note: The system can be scaled up or down according to the scale, and different PCR reactions need to be optimized; just follow the experimental plan.
4. The advantages and disadvantages of ordinary PCR
Advantages: classic methods, complete international and domestic standards; low cost of instrument reagents, PCR products can be recovered for other molecular biology experiments.
Disadvantages: easy to contaminate, cumbersome operation, only qualitative analysis, moderate sensitivity, non-specific amplification, when the non-specific band is the same size as the target band, it cannot be distinguished.
5. Scope of application
PCR is a molecular biology technology used to amplify specific DNA fragments. It can be regarded as a special DNA replication in vitro. The biggest feature is that it can increase a small amount of DNA by a large margin. Therefore, whether it is the remains of ancient creatures or historical figures in the fossils, as long as a little DNA can be isolated, it can be amplified by PCR for comparison.
6. Comparison of ordinary PCR instrument and qPCR instrument
A fluorescent signal acquisition system and a computer analysis and processing system are added on the basis of an ordinary PCR instrument to become a fluorescent quantitative PCR instrument. Its amplification principle is the same as that of an ordinary PCR instrument, except that the primers added during PCR amplification are used Isotopes, fluorescein, etc. are labeled, and primers and fluorescent probes are used to specifically bind to the template at the same time for amplification. The results of the amplification are collected by the fluorescence signal acquisition system in real time, and the signal is connected and transported to the computer analysis and processing system to obtain a quantitative real-time result output.
Note: Sensitivity refers to the minimum value that the PCR amplification reaction can detect the target gene. Specificity refers to the ability to specifically amplify the target fragment rather than other fragments during the PCR amplification process.
7. Common problems and countermeasures of PCR experiments
Question 1: The positive control has a band, but the sample has no result
Reasons: ①The template contains inhibitors and the content is low; ②Buffer is not suitable for the sample; ③Primers are improperly designed or degraded; ④Reaction conditions: the annealing temperature is too high and the extension time is too short.
Countermeasures: ①Purify the template or use the kit to extract template DNA or increase the amount of template; ②Change the buffer or adjust the concentration; ③Redesign the primers (avoid inter-chain dimer and intra-chain secondary structure) or replace a new tube of primers ④Reduce the annealing temperature and extend the extension time.
Question 2: Non-specific amplification
Reasons: ①Poor primer specificity; ②The template or primer concentration is too high; ③The amount of enzyme is too much; ④The Mg2+ concentration is too high.
Countermeasures: ①redesign primers or use nested PCR; ②appropriately reduce the template or primer concentration; ③appropriately reduce the amount of enzyme; ④reduce the Mg2+ concentration, or change the mix.
Question 3: GC-rich (normal range is 40-60%, >60% is GC-rich) area is difficult to amplify
Reason: The GC-rich region needs a higher temperature to open the double strands, and the melting may be incomplete at the conventional denaturation temperature; at the same time, the G+C rich region of the single strand is prone to self-complementary pairing to form a stable hairpin secondary structure. It is difficult for PCR primers to bind to the template, and it is also difficult for DNA polymerase to extend or stop the extension. As a result, serious non-specific bands appear and even target genes cannot be amplified.
Countermeasures: ①Increase the pre-denaturation temperature or time to fully open the double-strand; ②TD-PCR (gradient PCR); ③Change to hot-start enzyme or mix; ④Add co-solvents such as DMSO to assist DNA denaturation and reduce the Tm value of primers. Improve the specificity of product amplification.
Question 4: The product is in a smear state on the gel
Reasons: ①The template is impure; ②The buffer is not suitable; ③The annealing temperature is too low; ④The amount of enzyme is too much; ⑤The concentration of dNTPs and Mg2+ is too high.
Countermeasures: ①Purify the template; ②Replace Buffer or mix; ③Properly increase the annealing temperature; ④Set the amount of enzyme added according to the concentration ratio; ⑤Appropriately reduce the dNTPs and Mg2+ concentration; ⑥Reduce the number of reaction cycles.
Question 5: The target amplification product appears in the blank control
Reason: Cross-contamination of target sequence or amplified product.
Countermeasures: ①Operation should be careful and gentle to prevent the target sequence from being sucked into the sample gun or spilled out of the centrifuge tube; ②Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be autoclaved. All centrifuge tubes and sample pipette tips should be used for one time; ③All reagents are best to be aliquoted first, and then stored at low temperature.
dPCR
In 1999, Bert Vogelstein and Kenneth W. Kin-zler formally proposed the concept of digital PCR. dPCR is an absolute quantification technology of nucleic acid molecules. Compared with qPCR, dPCR does not depend on the cycle Ct value of the amplification curve and is not affected by the amplification efficiency. It can directly read the number of DNA molecules and can absolutely quantify the nucleic acid molecules of the starting sample. The number of segmentation units, the number of fluorescence channels, the complexity of operations, and the risk of contamination are important indicators for evaluating digital PCR platforms. In addition, the use of multiple digital PCR platforms to verify each other and the use of accurate reference materials is the main method for evaluating digital PCR platforms.
1. What is dPCR
dPCR: digital PCR (digital PCR) is an absolute quantification technology for nucleic acid molecules based on the Poisson distribution principle. By limiting the dilution of the sample to be tested, so that there is only one copy or no target DNA molecule in each reaction chamber, and then adding a fluorescent signal for PCR amplification, mutations as low as one copy can be detected.
2. The basic principle 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.
3. The advantages and disadvantages of dPCR
Advantages: Realize absolute quantification, higher sensitivity and specificity, can detect low-copy samples, simple operation process and result analysis.
Disadvantages: The equipment and reagents are expensive, the operation is complicated, the detection time is long, the cost is high, and the detection range is narrow. This method is suitable for detecting low-frequency mutations in ctDNA or other low-concentration samples, especially for "liquid biopsy".
4. Main purpose
Gene expression: It can detect subtle changes with high accuracy and good repeatability. Mutation detection: It can detect low-content mutation genes, and the sensitivity of mutation sequence detection is high. Copy number variation CNV detection: The copy number of the target gene can be obtained by accurately measuring the target gene and the reference gene, and calculating the ratio. Verification of second-generation sequencing data: Absolute quantitative analysis can be directly performed on the results of second-generation sequencing data.
5. Compared with conventional PCR methods, the advantages of digital PCR
① Absolute quantification can be achieved: Routine PCR quantification requires the development of a standard DNA curve with known copy number, but because the sample to be tested and the standard curve are not in a unified system, there will be differences in conditions, and the difference in PCR amplification efficiency will affect quantification The accuracy of the results. The digital PCR is not affected by the standard curve and amplification kinetics, and can be used for absolute quantification. ②Low sample demand: suitable for precious samples or samples with serious nucleic acid degradation. ③High sensitivity: Digital PCR divides the traditional PCR reaction system into tens of thousands of independent PCR reactions. These reactions can accurately detect small differences in target fragments, single copies or even low-concentration mixed samples. And can avoid the formation of non-homologous heteroduplex. ④High tolerance: Since the target sequence is allocated to multiple independent reaction systems, the interference of background signals and inhibitors on the reaction is significantly reduced, and the amplification matrix effect is greatly reduced.
6. The mainstream digital PCR in the market
In 2013, dPCR was rated as one of the top ten breakthrough technologies by MIT Science and Technology Review. In 2017, it was selected as one of the top ten emerging technologies in the world by the World Economic Forum. After several years of development, it is currently a clinical molecule in China Testing has been applied. The mainstream dPCR currently on the market includes: ①Life Technologies 3D digital PCR (chip-based digital PCR): It can be considered as microporous digital PCR, which is mainly to disperse 20 ul reaction system into 20,000 micropores for reaction, turning into 20,000 reaction systems After the PCR reaction is over, use CCD to take pictures and count the positive reaction wells. ②Bio-Rad droplet digital PCR (water-in-oil droplet digital PCR): A 20ul reaction system is used in a droplet reactor to form 20,000 water-in-oil reaction systems. After the PCR reaction is completed, the principle of flow cytometry can be used. Detect each liquid and count the number of droplets that have a positive reaction. ③Rain Dance's digital PCR (water-in-oil droplet digital PCR): The principle is the same as the Bole microdrop digital PCR, but its liquid forming ability is much stronger than that of Bole, and theoretically it can generate 10 million small oil droplets. Among them, Rain Dance has the highest price, Bio-Rad second, and Life Technologies has the lowest price.
Technological advances have continuously injected new vitality into PCR technology, making nucleic acid detection more convenient and more accurate. At present, the three generations of PCR technology have their own advantages and disadvantages, and each has different application fields. There is no relationship between one generation and the other. Researchers can choose the appropriate PCR technology according to their actual conditions!
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