Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as a special kind of DNA replication in vitro. DNA polymerase I was first discovered in 1955, and the Klenow fragment of Escherichia coli with more experimental value and practicality was discovered by Dr. H. Klenow in the early 1970s. However, because this enzyme is not resistant to high temperature and can be denatured at high temperature, it is not suitable for using high temperature denatured polymerase chain reaction. The enzyme used now (Taq polymerase for short) was isolated from the thermophilic bacteria in hot springs in 1976. Because of its high temperature tolerance, it is an ideal enzyme, but it has been widely used since 1980s. The original prototype concept of PCR is similar gene repair and replication, which was put forward by Dr. Kjell Kleppe in 197 1. He published the first simple and short-lived gene replication experiment (similar to the first two cycles of PCR). The PCR developed today was developed by Dr. Kary B. Mullis in 1983. Dr. Mullis worked in PE company, so PE company has a special position in the field of PCR. Dr Muhlis published the first related paper with Saiki and others in 1985. Since then, the application of PCR has advanced by leaps and bounds, and the quality of related papers can be said to be unmatched by many other research methods. Subsequently, PCR technology was widely used in biological research and clinical application, and became the most important technology in molecular biology research. Muhlis also won the 1993 Nobel Prize in chemistry.

2 PCR principle

The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denaturation-annealing-extension:

(1) denaturation of template DNA: After the template DNA is heated to about 93℃ for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated, so as to combine with primers and prepare for the next round of reaction;

(2) Annealing (renaturation) of template DNA and primer: after the template DNA is denatured into single strand by heating, the temperature is reduced to about 55℃, and the primer and the complementary sequence of the template DNA single strand are paired and combined;

③ Primer extension: Under the action of TaqDNA polymerase, DNA template-primer conjugate takes dNTP as the reaction raw material and the target sequence as the template. According to the principle of base complementary pairing and semi-conservative replication, a new semi-conservative replication chain complementary to the template DNA chain can be synthesized, and more "semi-conservative replication chains" can be obtained by repeating the three processes of cyclic denaturation-annealing-extension, which can be used as the template for the next cycle. It takes 2 ~ 4 minutes to complete a cycle, and the target gene to be amplified can be amplified by a million times in 2 ~ 3 hours.

3 PCR reaction system and reaction conditions

3. 1 standard PCR reaction system

10× amplification buffer 10μl

200μl mixture of four dNTP.

Primer10 ~100μ l

Template DNA 0. 1 ~ 2 μ g

Taq DNA polymerase 2.5 microliters

Mg2+ 1.5 mmol/L.

Add double or triple distilled water100μ l.

3.2 Five elements of PCR reaction

There are five main substances involved in PCR reaction, namely primers (PCR primers are DNA fragments, and primers for DNA replication in cells are RNA strands), enzymes, dNTP, templates and buffers (Mg2+ is needed). [[PCR step]

The standard PCR process is divided into three steps:

1.DNA denaturation (90℃-96℃): Under the action of heat, the hydrogen bond of the double-stranded DNA template is broken to form single-stranded DNA.

2. Annealing (25℃-65℃): The temperature of the system decreases, and the primer and the DNA template combine to form a local double strand.

3. Extension (70℃-75℃): Under the action of Taq enzyme (about 72℃, the activity is the best), dNTP is used as raw material to extend from the 5' end to the 3' end of the primer to synthesize a DNA strand complementary to the template.

After denaturation, annealing and extension in each cycle, the DNA content doubles. At present, some PCR can be replicated in a short time even if the Taq enzyme activity is not optimal due to the short amplification region, so it can be changed to a two-step method, that is, annealing and extension are carried out at 60℃-65℃ at the same time, so as to reduce the heating and cooling process once and improve the reaction speed.

4 PCR reaction characteristics

4. 1 has strong specificity.

The specific determinants of PCR reaction are:

① Correct combination of specificity of primer and template DNA;

② the principle of base pairing;

③ The authenticity of ③Taq DNA polymerase synthesis reaction;

④ The specificity and conservation of the target gene.

The correct combination of primers and templates is the key. The combination of primer and template and the extension of primer chain follow the principle of base pairing. Because of the fidelity of polymerase synthesis reaction and the high temperature resistance of Taq DNA polymerase, the combination (renaturation) of template and primer in the reaction can be carried out at higher temperature, the specificity of combination is greatly increased, and the amplified target gene fragment can maintain high accuracy. By selecting highly specific and conservative target gene regions, its specificity will be higher.

4.2 High sensitivity

The amount of PCR products increases exponentially, and the initial template to be tested can be amplified to the level of μg=-6 according to Pique (pg= 10- 12). A target cell can be detected from 6.5438+0 million cells; In virus detection, the sensitivity of PCR can reach 3 RFU (plaque forming unit); In bacteriology, the lowest detection rate is 3 bacteria.

4.3 Simple and quick

Application of thermostable Taq DNA polymerase in PCR reaction. After the reaction solution is added at one time, denaturation-annealing-extension reaction is carried out in DNA amplification solution and water bath pot, and the amplification reaction is generally completed within 2 ~ 4 hours. Amplification products are generally analyzed by electrophoresis, and isotopes are not needed, so there is no radioactive pollution and it is easy to popularize.

4.4 The purity of the specimen is low.

There is no need to separate viruses or bacteria from cultured cells, and crude DNA and RNA can be used as amplification templates. It can be directly used for DNA amplification and detection of clinical samples such as blood, body cavity fluid, mouthwash, hair, cells and living tissues.

5 PCR FAQ

5. 1 false negative, no amplification band.

The key links of PCR reaction are ① preparation of template nucleic acid, ② quality and specificity of primers, ③ quality of enzyme, and ④PCR cycle conditions. To find out the reason, we should also analyze and study the above links.

Template: ① template contains protein, ② template contains Taq enzyme inhibitor, ③ protein in template has not been eliminated, especially histone in chromosome, ④ template is lost too much during extraction and preparation, or phenol is inhaled. ⑤ The template nucleic acid is not completely denatured. When the quality of enzyme and primer is good, there is no amplification band, which is probably due to the failure of sample digestion and template nucleic acid extraction process. Therefore, in order to prepare effective and stable digestive juice, the procedure should be fixed and cannot be changed at will.

Inactivation of enzyme: it is necessary to replace the new enzyme or use the old and new enzymes at the same time, and analyze whether the false negative is caused by the loss or deficiency of enzyme activity. It should be noted that Taq enzyme or ethidium bromide is sometimes forgotten.

Primers: The quality of primers, the concentration of primers and whether the concentrations of two primers are symmetrical are the common reasons for the failure of PCR or the unsatisfactory amplification band, which is easy to disperse. The quality of primer synthesis in some batches is problematic. One of the two primers has high concentration and the other has low concentration, which leads to low efficiency of asymmetric amplification. The countermeasures are as follows: ① The selected primers are used to synthesize a single position. ② The concentration of primer depends not only on OD value, but also on agarose gel electrophoresis of primer stock solution. 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 the other primer has no band. At this time, PCR may fail, and it is necessary to negotiate with the primer synthesis unit. If one primer has high brightness and the other has low brightness, the concentration should be balanced when diluting the primer. (3) Primers should be stored in small packages with high concentration to avoid deterioration and degradation due to repeated freezing and thawing or long-term storage in the refrigerator. ④ The primer design is unreasonable. If the length of primers is not enough, dimers will be formed between primers.

Mg2+ concentration: Mg2+ ion concentration has great influence on PCR amplification efficiency. Too high concentration will reduce the specificity of PCR amplification, and too low concentration will affect the yield of PCR amplification, and even make PCR amplification fail without producing amplification bands.

Variation of reaction volume: Generally, the volumes used for PCR amplification are 20ul, 30ul and 50ul. Or 100ul, which volume to use for PCR amplification is set according to different purposes of scientific research and clinical testing. When making a small volume, such as 20ul, you must set the mode conditions, otherwise it is easy to fail.

Physical reasons: denaturation is very important for PCR amplification, such as low denaturation temperature and short denaturation time, which is easy to produce false negative; Too low annealing temperature will lead to non-specific amplification and reduce the efficiency of specific amplification. Too high annealing temperature will affect the combination of primers and templates and reduce the efficiency of PCR amplification. Sometimes it is necessary to use a standard thermometer to detect the denaturation, annealing and extension temperature of an amplifier or a water-soluble pot, which is also one of the reasons for the failure of PCR.

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

5.2 false positive

PCR amplification bands are consistent with the target sequence bands, and sometimes the bands are more neat and brighter.

Improper primer design: the selected amplification sequence has homology with the non-target amplification sequence, so the amplified PCR product is the non-target sequence when PCR amplification is carried out. If the target sequence is too short or the primer is too short, it is easy to appear false positive. Primers need to be redesigned.

Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: First, cross-contamination of whole genome or large fragments leads to false positives. This false positive can be solved by the following methods: the operation should be careful and gentle to prevent the target sequence from being sucked into the loading gun or splashed out of the centrifuge tube. Except for enzymes and substances that can't withstand high temperature, all reagents or equipment should be autoclaved. The centrifuge tube and injection gun head used should be used once. If necessary, before adding samples, the reaction tubes and reagents are irradiated with ultraviolet rays to destroy the existing nucleic acids. The second is the pollution of small fragments of nucleic acid in the air, which are shorter than the target sequence, but have certain homology. They can be spliced with each other, and after complementary with primers, PCR products can be amplified to produce false positives, which can be reduced or eliminated by nested PCR.

5.3 Non-specific amplification bands appear

The bands amplified by PCR are inconsistent with the expected size, large or small, or specific amplification bands and non-specific amplification bands appear at the same time. There are two reasons for the appearance of nonspecific bands: one is that the primers are not completely complementary to the target sequence, or the primers polymerize to form dimers. Secondly, too high Mg2+ ion concentration and too low annealing temperature are related to too many PCR cycles. Secondly, the quality and quantity of enzymes, often some enzymes from some sources are prone to non-specific bands while others are not, and sometimes non-specific amplification will occur when the amount of enzymes is too high. The countermeasures are as follows: redesign the guide if necessary. Reduce the amount of enzyme or replace the enzyme from other sources. Reduce the amount of primers, appropriately increase the amount of templates, and reduce the number of cycles. Appropriately increase the annealing temperature or adopt the double temperature point method (denaturation at 93℃, annealing extension at about 65℃).

5.4 Sheet traction belt or smear belt appears.

PCR amplification sometimes appears smear band or sheet band or carpet-like band. The reasons are often too many enzymes or poor enzyme quality, too high dNTP concentration, too high Mg2+ concentration, too low annealing temperature and too many cycles. The countermeasures are as follows: ① Reduce the amount of enzyme, or change the enzyme from other sources. ② Reduce the concentration of dNTP. ③ Reduce the concentration of Mg2+ appropriately. (4) Increase the amount of templates and reduce the number of cycles.