What is PCR (Polymerase Chain Reaction)?

1.1 Overview and principle of PCR technology

PCR (Polymerase Chain Reaction) is the polymerase chain reaction, which is catalyzed by DNA polymerase, using specific DNA as a template, using specific nucleic acid fragments, namely PCR primers, as the start and end points of amplification, through denaturation, annealing, and extension And other steps, the process of replicating a large number of daughter strand DNA complementary to the required DNA fragment in the parent strand template in an in vitro system.

The biggest feature of PCR is the specificity of the amplified product, the sensitivity of the amplification efficiency, and the simplicity of the amplification procedure. The primer sequence and its binding specificity with the template are the key to determining the result of the PCR reaction. PCR technology is a DNA synthesis amplification technology in vitro. It has the characteristics of high amplification efficiency, high amplification specificity, and simple and mature amplification system and technology. It is the most widely used technology in biology-related laboratories today.

1.1.1 Cycle steps of PCR

The PCR reaction process is actually a temperature cycle change process, generally including denaturation-annealing-extension three basic reaction steps, the basic steps are as follows:

(1) Denaturation of template DNA: The template DNA is heated to above 92～96℃ for 1～3 minutes to dissociate the template DNA double-stranded or the double-stranded DNA formed by PCR amplification and make it single-stranded. Combine with primers to prepare for the next round of reaction; although the denaturation time is determined by many factors, such as the complexity of template DNA, the geometry of the reaction tube, the type of PCR instrument and even the volume of the reaction, in actual experience, incubation at 94°C for 3 minutes is sufficient. Can meet the needs of most reactions. In addition, for DNA templates with high GC content, adding glycerol, extending the time, and applying nucleic acid analogs can increase the yield of PCR products.

(2) Annealing of template DNA and primers (renaturation): After the template DNA is denatured into single strands by heating, it is incubated at 37-65°C for 0.5 to 1 minute, and the oligonucleotide primers are paired and combined with the complementary sequence of the template DNA single-stranded; The temperature of this step depends on the degree of homology between the primer and the target sequence and the base composition of the oligonucleotide primer. Since the number of moles of primers is absolutely greater than that of the target sequence, their pairing speed with the complementary sequence in the reaction is several orders of magnitude faster than the re-pairing between the template double strands.

(3) Primer extension: DNA template—primer conjugate system is extended and incubated at 68～72℃ for the corresponding time (the specific temperature of this step depends on the type of thermostable DNA polymerase, and the incubation time is based on the DNA polymerase Based on the efficiency, it depends on the length of the reaction product. For example, the most commonly used DNA polymerase Taq can amplify DNA fragments with a length of about 1Kb per minute at 72℃. Therefore, when amplifying two 2Kb DNA fragments The heat preservation temperature of this step should be set to 72℃ and the time is 2 minutes). Under the action of DNA polymerase, use dNTP as the reaction material and the target sequence as the template. According to the principle of base pairing and semi-reserved replication, a new one is synthesized. The half-reserved replication strand complementary to the template DNA strand.

Repeat the cycle of denaturation—annealing—extending the three processes, you can get more “semi-reserved replicated strands”, and this new strand can become the template for the next cycle, making the purpose of new amplification in each cycle The number of fragments grows exponentially. After a certain number of cycles, the number of target DNA fragments to be amplified will be amplified several million times. The number of cycles required to reach the plateau depends on the copy of the template in the sample.

1.1.2 General PCR reaction system

The components of the PCR reaction generally include: (1) template DNA; (2) primers; (3) four kinds of deoxyribonucleotides; (4) DNA polymerase; (5) reaction buffer, Mg2+, etc. At present, PCR reactions have all been carried out in PCR machines, and commercial PCR kits are generally used for PCR reactions. Therefore, generally researchers only need to prepare their own template DNA and PCR primers, and then operate according to the product instructions; The understanding of the various components of the PCR system is the basis for PCR experiments, especially when the amplification results are not ideal, the experiments can be improved accordingly to obtain the amplification results that meet the experimental needs.

General PCR reaction system mainly includes the following components:

(1) Template: PCR does not require very high template DNA purity. Generally, samples prepared by standard molecular biology methods can be used directly as templates without special treatment, but should try not to contain impurities that can inhibit the PCR reaction. , Such as proteases, nucleases, polymerase inhibitors, and proteins that can bind to DNA. It should be pointed out that the amount of template DNA should not be too high, otherwise the amplification may not be successful. In this case, the template can be diluted appropriately. In addition, when small fragments of DNA such as plasmids and pre-amplified fragments are used as templates, the amplification efficiency will be significantly higher than that of genomic DNA as a template. Therefore, when using small fragments of DNA as a template, minimize the amount of template. 10-9g to 10-12g level is enough.

(2) Primer: The final concentration of the primer in the PCR reaction is generally 1μmol/L. Too high a concentration will cause mismatch and non-specific amplification, and too low a concentration will result in no product or low yield. The dry powdered primers synthesized by commercial companies can generally be stored in ddH2O or dissolved at 100μmol/L, stored at -20℃, and can be added to the system according to the proportion during use.

(3) Substrates (dNTPs): dNTPs have strong acidity. The storage solution is adjusted to pH 7.0-7.5 with NaOH, and the general storage concentration is 10mmol/L. The components are prepared in equivalent amounts, and the final concentration of the reaction is 20-200μmol. /L. High concentration can speed up the reaction, but at the same time increase the error incorporation and experimental cost; low concentration can improve the accuracy, but the reaction speed will be reduced. It should be noted that changes in the concentration of dNTPs will affect the effective Mg2+ concentration, so if the concentration of dNTPs increases, the Mg2+ concentration should also increase.

(4) Magnesium ion: Mg2+ can affect the specificity of the reaction and the yield of PCR. The working concentration of Mg2+ is 1.5～2.0mM, and its corresponding dNTP is 200μmol/L. Taking Taq enzyme as an example, since dNTP competes with Taq enzyme for Mg2+, when the dNTP concentration reaches 1mmol/L, it will inhibit Taq enzyme activity. The use of magnesium ions is strict, and its effect is better than that of manganese ions, while calcium ions are ineffective.

(5) No Mg2+buffer: mainly composed of H2O, KCl, and Tris. Tris is used to control the pH value in the reaction system to ensure the reactivity of DNA polymerase; KCl not more than 50mM can lower the annealing temperature, but too high KCl will inhibit the activity of DNA polymerase.

(6) DNA polymerase: DNA polymerase is the guarantee for PCR amplification. The current PCR technology uses thermostable enzymes, which avoids the cumbersome need to add enzymes after each denaturation in the initial application of PCR technology. Work. At present, the most commonly used Taq enzyme can withstand the high temperature of 95 ℃ without inactivation, and its optimum pH is 8.3 to 8.5, the optimum temperature is 75 to 80 ℃, and 72 ℃ is generally used. It can catalyze the synthesis of a new DNA sub-strand complementary to the template DNA by linking dNTP molecules to the 3’end of the primer one by one in the direction of 5’→3′ on the basis of the principle of base complementation, using a single DNA strand as a template. The disadvantage of Taq enzyme is that there is no 3’→5′ exonuclease activity, so there is no correction function. A certain dNTP or Mg2 concentration is too high, which will increase its mismatch rate. Therefore, it is recommended to use 3 in a precision PCR laboratory. High-fidelity polymerase with exonuclease activity of’→5′. The amount of polymerase is generally added according to the recommended amount in the kit instructions.

In addition to the above standard components, some researchers or kits recommend adding gelatin, TritonX-100 or BSA (calf serum albumin) to the system to increase the stability of the enzyme. The possible reason is that they play a role in reducing the polymerization of the PCR tube. Adsorption of enzymes to make polymerase participate in the reaction as much as possible.

In addition, glycerol, dimethyl sulfoxide, and formamide can improve the specificity. These components may reduce the melting and chain separation temperature to make the template denaturation easier and improve the specificity of primer annealing to achieve the purpose of improving the specificity of the PCR reaction.

1.2 Primer design

1.2.1 The general idea of PCR primer design

One of the keys to the successful amplification of the PCR reaction is the correct design of the primers. Especially under the condition that there are a large number of PCR kits to avoid the complicated exploration of the experimental system and conditions by the researchers, the researchers only need to prepare the template and primers by themselves. Can.

The core idea of primer design is to strike a balance between amplification specificity and efficiency. Specificity refers to the frequency with which non-target fragments are amplified; amplification efficiency refers to how close the yield of the amplified product caused by a pair of primers in each cycle should be theoretically.

At present, there are a large number of primer design software available on the market. These software basically consider the quality of primers based on the following points: it may cause false positive amplification of non-target sequences, the hairpin structure of the primer itself, the formation of primer dimers, etc., false positive amplification The increase is related to the specificity of the primer, and the hairpin structure of the primer and the formation of the dimer have a greater impact on the amplification efficiency.

1.2.2 The composition of primers

The main body of the primer is usually a short 5’→3′ oligonucleotide chain that is strictly complementary to the target sequence. According to the different purposes of the experiment, a restriction enzyme recognition site is often added to the 5’of the primer. PCR irrelevant sequences or other various modifications, these modifications usually do not affect the first normal annealing and amplification of the primers, and in the subsequent cycles, because there are complementary templates, the system can also be amplified normally.

1.2.3 Several factors that should be considered when designing primers

(1) Primer length: The specificity of PCR amplification is controlled by the length of the primer and the annealing temperature. When the annealing temperature of the reaction is close to the Tm value, primers with a length of 18 to 24 bases have excellent specificity.

Theoretically, it is believed that each additional base on the primer will increase the specificity by a factor of 4, so the longer the primer, the more the number of bases, the more specific the specificity, but the longer the primer, the less template that can be primed during annealing. After the exponential amplification period is amplified, the amplified products will be significantly reduced. Therefore, if there is no special need, the complementary sequence part of the general primer does not need to exceed 25 bases.

The shorter the primer, the higher the probability that it will bind to the target sequence during annealing, so too few bases in the primer that are complementary to the target sequence will cause unnecessary non-target amplification products.

(2) GC% and Tm value: Tm value refers to the melting temperature of the duplex that binds the primer and the template. A lower Tm value results in the loss of primer specificity, in which case a large number of non-specific amplification products will appear. The Tm=2(A+T)+4(C+G) formula proposed by Suggs in 1981 has been praised by the majority of researchers due to its simplicity and relative accuracy. For a primer of about 20 bases, based on the above formula, it can be found that when the CG% is about 50%, the Tm value is about 56℃～62℃, which is more suitable for PCR reaction. Therefore, when designing primers, you should choose (A +T) and (C+G) approximately the same number of sequences. In particular, attention should be paid to make the Tm values of the upstream and downstream primers as close as possible. Ideally, the difference is within 2 to 3°C. This can make the upstream and downstream primers bind to the target sequence at the same efficiency during annealing in the PCR reaction, so as to obtain a stable and Sufficient amount of amplified product.

It should be pointed out that when setting the annealing temperature for PCR reaction, whether it is calculated by formula or predicted by software or the Tm value provided by the primer synthesis company, it can only be used for reference in fact. The actual annealing temperature of each PCR reaction is theoretically They are all unknown. Usually the annealing temperature is set to the reference Tm value + 2 or 4°C during the first amplification, and then adjusted according to the amplification results.

(3) The position of the primer in the target sequence: In addition to the specific and precise target fragment, in most experiments, the length of the PCR reaction product can be appropriately selected within a certain range, according to the purpose of the PCR product and the template The characteristics of the polymerase and the characteristics of the polymerase used can choose the appropriate product length. The PCR reaction in the general experiment is usually between 150 and 1000 bp, and if it is only to check a specific sequence, only 120 to 300 bp is sufficient. .

When selecting primers on the target sequence, pay attention to avoiding the region of single bases in a string, avoiding continuous CG sequences, and try to choose four regions with a random distribution of bases, so as to reduce the chance of primer homology and avoid Unnecessary consumption of primers in the amplification process; at the same time, there should be no complementary strands between the upstream and downstream primers, and there should be no homology with non-target amplified regions. In addition, pay special attention to the sequence at the 3’end. If it does not have a specific effect, this sequence must be strictly paired with the template DNA. It is best to use A, C, G for the last base, because T mismatches can also cause chain extension.

1.3 The specificity, efficiency and fidelity of PCR

The specificity, efficiency and fidelity of PCR are three important indicators that reflect the quality of a PCR experiment. Specific refers to a PCR reaction that produces only one amplified product, that is, the expected target sequence. Efficiency refers to how close the actual amount of product amplified in each cycle should be in theory. Faithfulness means that the mismatch induced by the DNA polymerase is negligible during the PCR reaction.

Each of these three parameters is affected by many components in the PCR reaction, including buffer systems, enzymes, templates, and even PCR cycle procedures, but the most regrettable thing is that the highly specific reaction conditions are not consistent with the high-yield reaction conditions At the same time, high fidelity often leads to low yields. Therefore, when designing PCR experiments, these three parameters should be optimized according to the purpose of the experiment. For example, in the analysis of fragment length polymorphism, the yield and specificity of PCR products are more important than fidelity; and if you are studying individual DNA molecules or rare mutations, the importance of fidelity is self-evident.

Specificity is the basis of PCR, and it first depends on the precise complementation of the primer design and the template. An ideal set of primers can effectively hybridize with the target sequence, while the hybridization with other sequences appearing in the template is negligible. Generally, the 4 or less consecutive base complementation between the primer and the non-target sequence in the template will not be reflected in the electrophoresis.

The efficiency of the PCR reaction affects the accumulation of specific products. According to the function relationship between the accumulation of the target sequence and the number of cycles given by Chien et al., the period of the highest amplification efficiency is the exponential growth period before 29 cycles, and the period of each cycle thereafter The efficiency begins to decrease greatly, and the product stops accumulating exponentially. At this time, theoretically, the number of copies of the target sequence has reached 1012, and the specific sequence of 1012 copies has met the requirements for most molecular biology experiments, and many applications of PCR are especially quantitative. All experiments require amplification to be completed in the exponential growth phase. Therefore, the general PCR reaction is taken out before 29 cycles, and there is no need to increase the number of cycles.

The fidelity of the PCR reaction is mainly related to enzymes. Although the fidelity can be greatly improved by changing the conditions of the reaction buffer, it is far inferior to the effect of replacing the high-fidelity polymerase. The high fidelity ability of polymerase is reflected in its 3’→5′ exonuclease correction activity. It should be pointed out that whether high-fidelity polymerase is used or not, the PCR process may have wrong base infiltration. Therefore, in experiments that require fidelity, the sequence obtained by PCR must be sequenced and verified.

1.4 Optimization of PCR

In PCR experiments, the most obvious pair of contradictions is that a large number of uncertain and unwanted products are generated during amplification, or no products are amplified. The essence of this contradiction is the conditions required for high specificity and high efficiency of PCR. Inconsistent.

The following are some conditions that are conducive to increasing the specificity of PCR. Adjusting in the opposite direction can increase the amplification efficiency, but it will reduce the specificity. Therefore, the best goal is to find a balance between the two directions.

As mentioned above, there are usually many conditions that affect a PCR reaction. Therefore, in actual operation, it will be time-consuming and labor-intensive to perform a full matrix analysis to find the best conditions. Therefore, the research process can only be adjusted for the relevant main variables. In the case of focusing on the primer and template pairing and the fidelity of the enzyme, the Mg2+ concentration and annealing temperature are the most significant factors that affect the amplification, so a simple small matrix analysis can be performed only for these two variables. Landing PCR is essentially a method to find an effective annealing temperature in a series of gradient temperatures and to appropriately ensure specificity.

1.5 PCR FAQ

Electrophoresis detection and storage of PCR products

It is best to test immediately after PCR, generally not more than 48 hours; the product is stored at -20°C for later use, or stored after purification.

False negative, no amplified band

The key steps of the PCR reaction are: (1) Preparation of template nucleic acid; (2) Quality and specificity of primers; (3) Quality and quantity of enzymes; (4) PCR cycling conditions. Finding the reasons should also be analyzed and researched for the above links.

Template: (1) The template contains miscellaneous protein; (2) The template contains Taq enzyme inhibitor; (3) The protein in the template is not digested and removed, especially the histones in the chromosome; (4) Lost during the extraction and preparation of the template Too much, or inhaled phenol; (5) The template nucleic acid is not completely denatured. When the quality of the enzymes and primers is good, no amplified bands will appear. It is most likely that the sample is digested, and the template nucleic acid extraction process is faulty. Therefore, it is necessary to prepare an effective and stable digestion solution. The program should also be fixed and should not be changed at will .

Enzyme inactivation: It is necessary to replace with a new enzyme, or use both the old and the new enzyme at the same time to analyze whether the enzyme activity is lost or not enough to cause false negatives. It should be noted that sometimes the Taq enzyme is forgotten.

Primers: The quality of primers, the concentration of primers, and whether the concentrations of the two primers are symmetrical are common reasons for PCR failure or unsatisfactory amplified bands and easy diffusion. The synthesis quality of primers of some batches is problematic. One of the two primers has a high concentration and one has a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: (1) select a good primer synthesis unit; (2) primer concentration Not only depends on the OD value, but also pay attention to the primer stock solution for agarose gel electrophoresis. There must be primer bands, and the brightness of the two primer bands should be roughly the same. For example, one primer has a band and one primer has no band. When doing PCR may fail, it should be resolved through consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance its concentration when diluting the primer; (3) The primer should be stored in small amounts at high concentration to prevent multiple freeze-thaw or long-term storage in the refrigerator, which may lead to deterioration and degradation of the primer; (4) The primer design is unreasonable, such as the length of the primer is not enough, the formation of dimers between the primers, etc.

Mg2+ concentration: Mg2+ ion concentration has a great influence on the efficiency of PCR amplification. Too high concentration can reduce the specificity of PCR amplification, while too low concentration will affect PCR amplification yield or even make PCR amplification fail without showing amplified bands.

Change of reaction volume: Usually the volume used for PCR amplification is 20μl, 30μl, 50μl, or 100μl. The volume to be used for PCR amplification is set according to the different purposes of scientific research and clinical testing. After making a small volume such as 20μl , When making a large volume, the conditions must be modelled, otherwise it is easy to fail.

Physical reason: Denaturation is very important for PCR amplification. For example, if the denaturation temperature is low, the denaturation time is short, and false negatives are very likely. The annealing temperature is too low, which can cause non-specific amplification and reduce the specific amplification efficiency. The annealing temperature is too high. Highly affect the combination of primer and template and reduce the efficiency of PCR amplification. Sometimes it is necessary to use a standard thermometer to check the denaturation, annealing and extension temperatures in the thermal cycler or the water bath. This is also one of the reasons for PCR failure.

Target sequence variation: If the target sequence is mutated or deleted, which affects the specific binding of the primer and the template, or the primer and template lose their complementary sequence due to the deletion of a certain segment of the target sequence, the PCR amplification will not be successful.

False positive, the amplified band is consistent with the target sequence band

Sometimes the PCR amplified bands are consistent with the target target sequence bands, and the bands are more orderly and brighter.

Inappropriate primer design: The selected amplified sequence has homology with the non-target amplified sequence. Therefore, the amplified PCR product is a non-targeted sequence during PCR amplification. If the target sequence is too short or the primer is too short, false positives are prone to occur. Need to redesign the primers.

Cross-contamination of target sequence or amplified product: There are two reasons for this type of contamination: (1) Cross-contamination of the entire genome or large fragments leads to false positives. This false positive can be solved by the following method: the operation should be careful and gentle to prevent the target sequence from being sucked into the sample gun or spilled out of the centrifuge tube. Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be autoclaved. All centrifuge tubes and sample injection tips should be used once. When necessary, the reaction tube and reagents are irradiated with ultraviolet rays before adding the specimens to destroy the existing nucleic acids. (2) The small fragments of nucleic acid in the air are contaminated. These small fragments are shorter than the target sequence but have a certain degree of homology. It can be spliced to each other, and after complementing the primers, PCR products can be amplified, which can lead to false positives. Nested PCR can be used to reduce or eliminate them.

Non-specific amplification band

The bands appearing after PCR amplification are inconsistent with the expected size, large or small, or both specific amplified bands and non-specific amplified bands appear at the same time. The reasons for the appearance of non-specific bands are: (1) The primer and the target sequence are not completely complementary, or the primer polymerizes to form a dimer. (2) The Mg2+ ion concentration is too high, the annealing temperature is too low, and the number of PCR cycles is too high. (3) The quality and quantity of enzymes. Often enzymes from some sources are prone to non-specific bands, but enzymes from another source do not appear. Too many enzymes sometimes cause non-specific amplification. The countermeasures are: redesign the primers when necessary. Reduce the amount of enzyme or switch to another source of enzyme. Reduce the amount of primers, increase the amount of template appropriately, and reduce the number of cycles. Properly increase the annealing temperature or use the two-temperature point method (93°C denaturation, 65°C annealing and extension).

Flaky drag or smear tape appears

In PCR amplification, smear bands, flaky bands, or carpet-like bands sometimes appear. The reason is often due to excessive amount of enzyme or poor quality of enzyme, too high dNTP concentration, too high Mg2+ concentration, too low annealing temperature, and too many cycles. The countermeasures are: (1) Reduce the amount of enzymes, or exchange enzymes from another source. (2) Reduce the concentration of dNTP. Appropriately reduce the Mg2+ concentration. Increase the amount of template and reduce the number of cycles.

[Main bibliography]

(1) C.W. Dieffenbacher, G.S. De Wexler, PCR technology experiment guide. Science Press: Beijing, 2000

(2) Sam Brook J, Fridge EF, Maniatis T. Molecular cloning experiment guide. second edition. Beijing: Science Press, 1998

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