What is a Polymerase Chain Reaction?

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify and amplify specific DNA fragments. It can be regarded as a special DNA replication in vitro. The biggest feature of PCR is that it can greatly increase the amount of DNA. . Therefore, whether it is the remains of paleontology, historical figures in the fossils, or the hair, skin or blood left by the murderer in the murders decades ago, as long as a tiny bit of DNA can be isolated, it can be amplified by PCR and compared Correct. This is where the power of "trace evidence" lies. The idea was first proposed by Mullis in the United States in 1983, and the polymerase chain reaction, the simple DNA amplification method, was invented in 1985, which meant the true birth of PCR technology. By 2013, PCR has developed to the third generation technology. In 1976, Chinese scientist Qian Jiayun discovered the stable Taq DNA polymerase and made a fundamental contribution to the development of PCR technology.

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify and amplify specific DNA fragments. It can be regarded as a special DNA replication in vitro. The biggest feature of PCR is that it can greatly increase the amount of DNA. . Therefore, whether it is the remains of paleontology, historical figures in the fossils, or the hair, skin or blood left by the murderer in the murders decades ago, as long as a tiny bit of DNA can be isolated, it can be amplified by PCR and compared. Correct. This is where the power of "trace evidence" lies. The idea was first proposed by Mullis in the United States in 1983, and the polymerase chain reaction, the simple DNA amplification method, was invented in 1985, which meant the true birth of PCR technology. By 2013, PCR has developed to the third generation technology. In 1976, Chinese scientist Qian Jiayun discovered the stable Taq DNA polymerase and made a fundamental contribution to the development of PCR technology.

PCR uses DNA to degenerate into single strands at high temperatures of 95 ° C in vitro. Primers and single strands are combined based on the principle of complementary base pairing at low temperatures (often around 60 ° C), and then the temperature is adjusted to the optimum level for the DNA polymerase. At the reaction temperature (around 72 ° C), the DNA polymerase synthesizes complementary strands in the direction from phosphate to five carbon sugars (5'-3 '). The PCR instrument based on polymerase is actually a temperature control device, which can well control between denaturation temperature, renaturation temperature, and extension temperature. ^[1]

Chinese name

Polymerase chain reaction

Foreign name

Polymerase Chain Reaction

Short name

PCR

Attributes

Chain reaction

current state

Third generation technology

proposer

Moulis ^[2]

PCR Creation of polymerase chain reaction PCR

Khorana (1971) and others first proposed the concept of in vitro nucleic acid amplification: "After DNA denaturation, hybridization with appropriate primers, extension of primers with DNA polymerase, and repeating this process can synthesize tRNA genes."

However, because the method of gene sequence analysis was not yet mature, heat-stable DNA polymerase was not reported, and the difficulty of primer synthesis, this idea seemed to have no practical significance. Coupled with the advent of molecular cloning technology to provide a way to clone and amplify genes, Khorana's vision was forgotten.

In 1985, Kary Mullis invented PCR while working at Cetus. Mullis had to synthesize DNA primers for sequencing, but was often troubled by not having enough template DNA.

On a Friday night in April 1983, while driving to a country house, he suddenly flashed the idea of "polymerase chain reaction".

In December 1983, Mullis saw the first PCR fragment of 49 bp in length after 10 cycles using isotope labeling;

The patent for PCR was applied for on October 25, 1985, and was approved on July 28, 1987 (Patent No. 4,683,202). Mullis was the first inventor;

Mullis was the co-author of the first PCR academic paper published in Science on December 20, 1985.

In May 1986, Mullis gave a special report at the Cold Spring Harbor Laboratory, and the world began to learn about PCR methods. ^[1]

PCR Principles of polymerase chain reaction PCR

The semi-reserved replication of DNA is an important way for biological evolution and passage. Double-stranded DNA can be denatured and unspooled into single-stranded DNA under the action of various enzymes. With the participation of DNA polymerase, it can be copied into the same two molecular copies according to the principle of complementary base pairing. It has been found in experiments that DNA can also undergo denaturation and melting at high temperatures, and can renaturate into double strands when the temperature decreases. Therefore, through temperature changes to control the denaturation and renaturation of DNA, the addition of design primers, DNA polymerase, dNTP can complete the replication of specific genes in vitro.

However, DNA polymerases can be inactivated at high temperatures. Therefore, a new DNA polymerase must be added for each cycle, which is cumbersome and expensive, which restricts the application and development of PCR technology.

The discovery of the thermostable DNA polymerase-Taq enzyme is a milestone for the application of PCR. The enzyme can withstand high temperatures above 90 ° C without inactivation, and does not need to add enzymes every cycle, making PCR technology very simple, At the same time, costs have been greatly reduced, and PCR technology has been widely used and gradually applied to the clinic.

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 of denaturation-annealing-extension: Denaturation of template DNA: After heating the template DNA to about 93 ° C for a certain time, dissociate the template DNA double-stranded DNA or double-stranded DNA formed by PCR amplification. Make it single-stranded so that it binds to the primer to prepare for the next round of reaction; Annealing (refolding) of the template DNA and the primer: After the template DNA is denatured by heating to a single-strand, the temperature drops to about 55 ° C. The complementary sequence of the single-stranded template DNA is paired and combined; Primer extension: The DNA template-primer conjugate uses dNTP as the reaction raw material and the target sequence as the template under the action of a DNA polymerase (such as TaqDNA polymerase) at 72 ° C. Based on the principle of complementary base pairing and semi-retention replication, a new semi-reserved replication strand complementary to the template DNA strand is synthesized. Repeating the three cycles of cyclic denaturation-annealing-extension can obtain more "semi-reserved replication strands". The new chain can become a template for the next cycle. It takes 2 to 4 minutes to complete each cycle, and the target gene to be expanded can be amplified several million times in 2 to 3 hours. ^[3]

Polymerase chain reaction preparation

PCR Polymerase chain reaction PCR reaction system

10 × amplification buffer	10l
4 dNTP mixtures (final concentration)	100 250mol / L each
Primer (final concentration)	5 20mol / L each
Template DNA	0.1 2g
Taq DNA polymerase	5 10 U
Mg 2+ (final concentration)	1 3mmol / L
Add double distilled water	100 l

The amount (or concentration) of dNTP, primer, template DNA, Taq DNA polymerase and Mg ²⁺ can be adjusted according to the experiment. The above table only provides approximate reference values.

Five elements of PCR reaction: There are mainly five kinds of materials participating in the PCR reaction: primers (PCR primers are DNA fragments, and primers for DNA replication in cells are an RNA strand), enzymes, dNTPs, templates, and buffers (of which Mg ^{2+ is} required ).

There are many ways to design primers, which are determined by the purpose of PCR in the experiment, but the basic principles are the same. There are two main sources of enzymes used in PCR: Taq and Pfu, which come from two different types of thermophages. Among them, Taq has high amplification efficiency but is prone to mismatches. Pfu amplification efficiency is weak but has error correction function. Therefore, in actual use, different choices must be made according to needs.

The template is the DNA used for amplification. It can be from any source, but there are two principles. The first purity must be high, and the second concentration must not be too high to avoid inhibition.

The composition of the buffer solution is the most complicated. Generally, it includes four active ingredients in addition to water: buffer system, generally using HEPES or MOPS buffer system; monovalent cations, generally using potassium ions, but ammonium ions can also be used in special circumstances; Valence cations, that is, magnesium ions, are determined according to the reaction system and do not need to be adjusted except in special cases; auxiliary components, such as DMSO, glycerol, etc., are mainly used to maintain enzyme activity and help DNA to unwind the structure. ^[3]

PCR Design of PCR primers for polymerase chain reaction

There are two primers in the PCR reaction, the 5 primer and the 3 primer. The primer is designed based on a single DNA strand (usually based on the information strand). The primer at the 5 end is the same as a small DNA sequence located at the 5 end of the fragment to be amplified. The primer at the 3 end is the same as the fragment to be amplified. A small DNA sequence at the 3 'end is complementary.

(1) Basic principles of primer design

• Primer length: 15-30bp, usually about 20bp.

• Primer bases: The G + C content is preferably 40-60%. Too little G + C is not effective for amplification. Too much G + C is prone to non-specific bands. ATGC is best distributed randomly, avoiding a series of 5 or more references to purine or pyrimidine nucleotides.

• There should be no complementary sequences within the primer.

• There should be no complementary sequence between the two primers, especially to avoid complementary overlap at the 3 'end.

• The homology between the primer and the sequence of the non-specific amplification region should not exceed 70%, and the 8 consecutive bases at the 3 end of the primer must not have a completely complementary sequence outside the region to be amplified, otherwise non-specific amplification may easily occur.

• The bases at the 3 'end of the primer, especially the last and penultimate bases, should be strictly matched. The best choice is G and C.

• The 5 'end of the primer can be modified. For example, a restriction enzyme site is added, a mutation site is introduced, a biotin, a fluorescent substance, a digoxin label is used, and other short sequences are added, including a start codon and a stop codon.

(2) Primer design software

• Primer Premier5.0 (Auto Search)

• vOligo6 (primer evaluation)

• vVector NTI Suit

• vDNAsis

• vOmiga

• vDNAstar

• vPrimer3 (online service)

Preparation of polymerase chain reaction template

The template for PCR can be either DNA or RNA.

The material of the template is mainly based on the amplification target of PCR. It can be pathogen samples such as viruses, bacteria, and fungi. It can also be a pathophysiological specimen such as cells, blood, amniotic fluid cells and the like. Forensic specimens include blood spots, fine spots, and hair.

The basic requirements for specimen processing are removal of impurities and partial purification of nucleic acids in the specimen. Most samples need to be treated with SDS and proteinase K. Difficult bacteria can be treated with lysozyme and EDTA. The obtained crude DNA was purified by extraction with phenol and chloroform, and then precipitated with ethanol as a template for a PCR reaction.

Control of polymerase chain reaction

• PCR reaction buffers provide the appropriate pH with certain ions

• The total concentration of magnesium ions should be higher than the concentration of dNTPs, usually 1.5mmol / L

• Substrate concentration dNTP is prepared at equimolar concentration, 20 200umol / L

• TaqDNA polymerase 2.5U (100ul)

• Primer concentration is generally 0.1 to 0.5umol / L

• Reaction temperature and number of cycles

Denaturation temperature and time: 95 , 30s

Annealing temperature and time are about 5 lower than the Tm value of the primer, generally 45 to 55

Extension temperature and time 72 , 1min / kb (within 10kb)

Tm value = 4 (G + C) +2 (A + T)

Number of cycles: generally 25 to 30 times. The number of cycles determines the yield of PCR amplification. The initial template concentration is low and the number of cycles can be increased to achieve an effective amount of amplification. But the number of cycles is not infinite. Generally, the number of cycles is about 30. After the number of cycles exceeds 30, the DNA polymerase activity gradually reaches saturation, and the amount of product no longer increases with the increase of the number of cycles, so-called "platform phase" occurs. ^[4]

Polymerase chain reaction cycle parameters

Polymerase chain reaction

Complete denaturation of the template DNA and full activation of the PCR enzyme are critical to the success of the PCR. It is recommended that the heating time refer to the reagent instructions. Generally, the activation time of the unmodified Taq enzyme is two minutes. ^[5]

Polymerase chain reaction denaturation step

Generally, the temperature of 95 ° C for 30 seconds is sufficient to completely denature various target DNA sequences, and this step can be shortened if possible. Too long denaturation damages enzyme activity, too short target sequence denatures incompletely, which easily leads to amplification failure. ^[5]

Polymerase chain reaction primer annealing

The annealing temperature needs to be determined from various aspects. Generally, the Tm value of the primer is used as a reference, and the annealing temperature is appropriately adjusted down according to the length of the amplification. Then make an estimate based on this experiment. The annealing temperature has a great influence on the specificity of PCR. ^[5]

Polymerase chain reaction primer extension

Primer extension is generally performed at 72 ° C (the optimal temperature for Taq enzymes). However, when the amplification length is short and the annealing temperature is high, this step can be omitted. The extension time depends on the length of the amplified fragment. Generally, it is recommended to be above 1000bp. The derivative containing Pfu and its derivative is set to 1min / kbp. ^[5]

Polymerase chain reaction cycle number

Most PCRs contain 25-35 cycles, and too many are prone to non-specific amplification. ^[5]

Polymerase chain reaction last extension

After the last cycle, the reaction was maintained at 72 ° C for 10-30 minutes. The primers are fully extended and the single-stranded product is annealed to double-stranded. ^[5]

Polymerase chain reaction step

The standard PCR process is divided into three steps:

DNA denaturation: (90 ° C-96 ° C): double-stranded DNA template under thermal action, hydrogen bonds are broken to form single-stranded DNA

Annealing: (60 ° C-65 ° C): The system temperature decreases, and the primers bind to the DNA template to form a local double strand.

Extension: (70 ° C-75 ° C): under the action of Taq enzyme (around 72 ° C, the best activity), using dNTP as the raw material, extending from the 3 end of the primer in a direction from 5 3 end, Synthesis of a DNA strand complementary to the template.

With each cycle of denaturation, annealing, and extension, the DNA content doubles. As shown in the figure: Now some PCRs can replicate in a short time because the amplification region is very short, even if the Taq enzyme activity is not optimal, so it can be changed to a two-step method, that is, annealing and extension at 60 ° C- It is carried out at 65 ° C to reduce the temperature rise and fall process once and improve the reaction speed. ^[5]

Polymerase chain reaction detection

The PCR reaction amplified a high number of copies, and the next step became critical. Fluorescein (ethidium bromide, EB) staining gel electrophoresis is the most commonly used detection method. The specificity of electrophoresis is not very high, so non-specific hybrids such as primer dimers can easily cause misjudgments. But because of its simplicity, it has become the mainstream detection method. In recent years, the detection methods represented by fluorescent probes have gradually replaced the electrophoresis method. ^[5]

Characteristics of polymerase chain reaction

Polymerase chain reaction

The specific determinants of the PCR reaction are:

Specific and correct binding of primers and template DNA;

Base pairing principle;

The fidelity of Taq DNA polymerase synthesis reaction;

Specificity and conservation of target genes.

Among them, the correct binding of primers and templates is the key. The binding of the primer to the template and the extension of the primer strand follow the principle of base pairing. The fidelity of the polymerase synthesis reaction and the high temperature resistance of TaqDNA polymerase allow the binding (refolding) of the template and primers in the reaction to be carried out at a higher temperature, the specificity of the binding is greatly increased, and the amplified target gene fragment It can also maintain high accuracy. By selecting the target gene region with high specificity and conservation, the degree of specificity is even higher.

High sensitivity in polymerase chain reaction

The amount of PCR product generated increases exponentially, and the starting test template on the order of picogram (pg = 10-12) can be amplified to the microgram (g = -6) level. One target cell can be detected from one million cells; the sensitivity of PCR in virus detection can reach 3 RFUs (plaque forming units); the minimum detection rate in bacteriology is 3 bacteria. ^[1]

Simple and fast polymerase chain reaction

The PCR reaction uses high temperature resistant Taq DNA polymerase. After the reaction solution is added once, the denaturing-annealing-extension reaction is performed on the DNA amplification solution and water bath, and the amplification reaction is generally completed in 2 to 4 hours. Amplification products are generally analyzed by electrophoresis, and it is not necessary to use isotopes. There is no radioactive contamination and it is easy to popularize.

Polymerase chain reaction requires low purity

There is no need to isolate viruses or bacteria and culture cells, crude DNA products and RNA can be used as amplification templates. Can be directly used in clinical specimens such as blood, body cavity fluid, cough fluid, hair, cells, living tissues and other DNA amplification tests. ^[1]

Frequently asked questions about polymerase chain reaction

The electrophoretic detection time of PCR products is generally within 48 hours, and some are better than the same day electrophoresis detection. After 48 hours, the band pattern is irregular or even disappears.

Polymerase chain reaction false negative

No amplified bands appear. The key steps of the PCR reaction are the preparation of template nucleic acid, the quality and specificity of primers, the quality of the enzyme and the use of ethidium bromide, and the conditions of PCR cycle. Finding the reason should also carry out analysis and research on the above links.

Templates: The template contains miscellaneous proteins, The template contains Taq enzyme inhibitors, The proteins in the template are not digested and removed, especially the histones in the chromosomes, Excessive loss during extraction of the template, or inhalation of phenol. The template nucleic acid is not completely denatured. When the quality of enzymes and primers is good, no amplification bands appear. It is most likely that the sample is digested. The template nucleic acid extraction process is defective. Therefore, an effective and stable digestion treatment solution must be prepared. The procedure should also be fixed and should not be changed at will. . ^[1]

Polymerase chain reaction negative

It should be noted that sometimes forget to add Taq enzyme or ethidium bromide. Primers: Primer quality, primer concentration, and whether the concentrations of the two primers are symmetrical are common causes of PCR failure or unsatisfactory amplification bands. Some batches have problems with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in a low-efficiency asymmetric amplification. The countermeasures are as follows: Select a good primer synthesis unit. Primer concentration depends not only on the OD value, but also on the agarose gel electrophoresis of the primer stock. Primer bands must be present, and the brightness of the two primer bands should be approximately the same. The band may fail in PCR at this time, and it should be resolved through consultation with the primer synthesis unit. For example, if one primer has high brightness and low brightness, the concentration should be balanced when diluting the primer. Primers should be stored in high concentrations and small quantities to prevent repeated freezing and thawing or long-term refrigerated parts of the refrigerator, which will cause primer deterioration.

Degradation failure. Primer design is not reasonable, such as insufficient primer length, dimer formation between primers, etc.

Mg ²⁺ concentration: Mg ²⁺ ion concentration has a great influence on the efficiency of PCR amplification. Too high concentration can reduce the specificity of PCR amplification. Too low concentration will affect the yield of PCR amplification and even make PCR amplification fail without expanding. Add strips.

Changes in reaction volume: The volumes used for PCR amplification are usually 20ul, 30ul, and 50ul. Or 100ul, how much 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, and then making a large volume, you must mold the conditions, otherwise it is easy to fail.

Physical reason: Denaturation is very important for PCR amplification, such as low denaturation temperature and short denaturation time, false negatives are likely to occur; too low annealing temperature can cause non-specific amplification and reduce specific amplification efficiency. High-impact primer binding to template reduces PCR amplification efficiency. Sometimes it is necessary to use a standard thermometer to test the denaturation, annealing, and extension temperatures in the amplicon or water-soluble pot, which is one of the reasons for the failure of PCR.

Target sequence mutation: If the target sequence is mutated or deleted, which affects the specific binding of the primer to the template, or the primer and template lose the complementary sequence due to the deletion of a certain segment of the target sequence, the PCR amplification will not be successful. ^[1]

Polymerase chain reaction false positive

The PCR amplification bands appear to be consistent with the target target sequence bands, and sometimes the bands are evener and brighter.

Primer design is not appropriate: The selected amplified sequence has homology with the non-target amplified sequence, so when PCR amplification is performed, the amplified PCR product is a non-target sequence. The target sequence is too short or the primers are too short, which is prone to false positives. Primers need to be redesigned.

Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: First, cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be solved by the following methods: Care should be taken during operation to prevent the target sequence from being sucked into the sample gun or splashed out of the centrifuge tube. With the exception of 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. If necessary, before adding the sample, the reaction tube and reagents are irradiated with ultraviolet rays to destroy the existing nucleic acids. The second is the nucleic acid pollution of small fragments in the air. These small fragments are shorter than the target sequence, but have certain homology. They can be spliced to each other, and after complementary to the primers, PCR products can be amplified, which leads to the generation of false positives, which can be reduced or eliminated by nested PCR.

Non-specific amplification bands appear:

The bands that appeared after PCR amplification were inconsistent with the expected size, larger or smaller, or both specific and non-specific amplification bands appeared. Non-specific bands appear due to the following reasons: First, the primers are not completely complementary to the target sequence, or the primers polymerize to form a dimer. The second is that the concentration of Mg ²⁺ is too high, the annealing temperature is too low, and the number of PCR cycles is too much. The second is the quality and quantity of enzymes. Often enzymes from some sources are prone to non-specific bands, while enzymes from other sources are not. A large amount of enzymes sometimes causes nonspecific amplification. The countermeasures are: redesign the primers if necessary. Reduce the amount of enzyme or swap the enzyme from another source. 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 (denaturation at 93 ° C, annealing and extension at about 65 ° C).

A flaky tow or smear appears:

Smear bands or sheet bands or carpet-like bands sometimes appear in PCR amplification. The reasons are usually caused by too much enzyme or poor quality of enzyme, too high dNTP concentration, too high Mg ²⁺ concentration, too low annealing temperature, and too many cycles. The countermeasures are: reduce the amount of enzyme, or swap the enzyme from another source. Reduce the concentration of dNTP. Reduce Mg ²⁺ concentration appropriately. Increase the number of templates and reduce the number of cycles. ^[3]

Clinical application of polymerase chain reaction

For the treatment of infectious diseases, tumors and genetic diseases. ^[6]

Polymerase chain reaction infectious disease

The most valuable application area of PCR in medical laboratory science is the diagnosis of infectious diseases. In theory, as long as a pathogen is present in the sample, PCR can detect it. Normal laboratories can also detect 10 to 100 copies of genes, while current pathogen antigen detection methods generally require 105-7 pathogens to be detected. The detection of pathogens by PCR solves the "window period" problem of immunological detection, and can determine whether the disease is in a recessive or subclinical state.

Data from quantitative PCR studies have shown that there is a correlation between the number of pathogens and the severity, infectivity, and treatment effect of infectious diseases. Many studies have shown that the length of incubation period and clinical symptoms after human immunodeficiency virus (HIV) infection are significantly related to the amount of virus in the blood; there have also been studies showing that when the HIV viral load is below a certain value, it is not infectious.

Quantitative studies of hepatitis B virus and hepatitis C virus have found that the amount of virus is related to the efficacy of certain drugs. For example, interferon therapy is not sensitive to those with high copies of the hepatitis virus, and sensitive to those with low copies; some drugs have the effect of significantly reducing the high copies of the virus. ^[6]

Polymerase chain reaction tumor

Increased oncogene expression and mutations can occur in the early and benign stages of many tumors. PCR technology can not only detect gene mutations effectively, but also accurately detect the expression of oncogenes, which can be used for early diagnosis, typing, staging and prognosis of tumors.

The formation of BCR / ABL fusion genes caused by proto-oncogene translocation can be detected in almost all patients with chronic myelogenous leukemia. Quantitative PCR technology can determine the number of trace residual malignant cells by detecting the expression of BCR / ABL fusion genes. Basis for treatment effect and estimated risk of relapse.

The carcinogenicity of some viruses is also related to viral load. FQ-PCR results of EB virus load have been used for early detection and follow-up of nasopharyngeal carcinoma. ^[6]

Polymerase chain reaction genetic disease

The first clinical application of PCR technology began with the detection of mutations in sickle cells and beta-thalassemia. Mutations and deletions of genes can cause imbalanced expression of various globin proteins. Detection of various globin gene expression differences by FQ-PCR is an effective method for the diagnosis of thalassemia. ^[6]

Polymerase chain reaction test contamination

The biggest feature of the PCR reaction is that it has large amplification capacity and extremely high sensitivity, but the headache is that it is easy to contaminate, and extremely small amounts of contamination can cause false positives. ^[6]

Causes of polymerase chain reaction contamination

1. Cross-contamination between specimens: The main contamination of specimens is the contamination of the container used to collect the specimens, or when the specimens are placed, they are not sealed tightly outside the container, or the specimens are adhered to the container, causing cross-contamination between them; During the process, contamination between specimens due to contamination of the suction gun; some microbial specimens, especially viruses, can spread with aerosols or aerosols, causing contamination between each other.

2. Contamination of PCR reagents: It is mainly due to the contamination of PCR nucleic acid template by the sample gun, container, double distilled water and other solutions during the preparation of PCR reagents.

3. PCR product contamination: This is the most common and most common contamination problem in PCR reactions. Because the PCR product has a large copy volume (generally 1013 copies / ml), which is much higher than the limit of several copies detected by PCR, a very small amount of PCR product contamination can form a false positive. Another form that is easy to overlook is that aerosol contamination is the most likely form of contamination of PCR products. Aerosols can be formed when the air and liquid are rubbed, and the reaction tube is shaken more violently during operation. Aerosols can be formed and contaminated when the lid is opened, when the sample is sucked, and when the sample is contaminated by the sampling gun. It is calculated that an aerosol particle can contain 48,000 copies, so the pollution caused by it is a problem that deserves special attention.

4. Contamination of cloned plasmids in laboratories: This problem is also common in molecular biology laboratories and some laboratories using cloned plasmids as a positive control. Because the cloning plasmid content is relatively high in a unit volume, in addition, more tools and reagents are required in the purification process, and the plasmid in living cells has a strong vitality due to the simplicity of growth and reproduction of living cells. The possibility of pollution is also high. ^[6]

Monitoring of polymerase chain reaction contamination

A good laboratory should always pay attention to the monitoring of pollution and consider whether it is the cause of pollution in order to take measures to prevent and eliminate pollution.

1. Positive control: A PCR positive control should be set up in the establishment of a PCR reaction laboratory and general inspection units. It is an important reference mark for the success of the PCR reaction and the position and size of the product bands in accordance with the theoretical requirements. The positive control should be selected from the samples with medium amplification and good reproducibility, and the product is identified by various identifications. If the recombinant plasmid is used as a positive control, its content should be low or high (less than 100 copies). However, positive controls, especially recombinant plasmids and high-concentration positive specimens, are highly likely to contaminate detection or amplification samples. Therefore, when a certain PCR reagent is stable by its own use and the inspector knows it, the positive control may be omitted in the subsequent experiments.

2. Negative control: Be sure to make a negative control for each PCR experiment. it includes:

(1) Specimen control: If the specimen to be tested is serum, the normal serum after identification is used as a control; if the specimen to be tested is tissue cells, the corresponding tissue cells are used as a control.

(2) Reagent control: No template DNA or RNA is added to the PCR reagent, and PCR amplification is performed to monitor whether the reagent is contaminated.

3. Repeatability test.

4. Select primers from different regions for PCR amplification. ^[6]