Polymerase chain reaction (PCR) is used to amplify specific DNA fragmentsMolecular Biology TechnologyIt 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 and historical figures in fossils, or the hair, skin or blood left by the murderer in the murder case decades ago, as long as a little bit ofDNACan be amplified by PCR for comparison.This is also the power of "micro evidence".In 1983, Mullis of the United States first proposed the idea. In 1985, he invented the polymerase chain reaction, that is, the simple DNA amplification method, which means the real birth of PCR technology.By 2013, PCR has developed into the third generation technology.In 1976, Chinese scientist Qian Jiayun discovered a stable Taq DNA polymerase, which also made a fundamental contribution to the development of PCR technology.
PCR usesDNAIn vitro, at 95 ° C high temperature, the primer will become a single chain. At low temperature (usually around 60 ° C)BaseThe principle of complementary pairing is combined and readjustedtemperatureTo the optimal reaction temperature of DNA polymerase (about 72 ° C), DNApolymeraseComplementary chains are synthesized along the direction from phosphoric acid to pentose (5 '- 3').The PCR instrument based on polymerase is actually a temperature control device, which can well control the denaturation temperature, renaturation temperature and extension temperature.[1]
Khorana (1971) and others first proposednucleic acidThe idea of in vitro amplification:DNA denaturation, and appropriate primershybridization, usingDNA PolymeraseThe tRNA gene can be synthesized by extending the primer and repeating the process continuously. "
However, because the gene sequence analysis method was not yet mature, the heat stable DNA polymerase had not been reported, and the primer synthesis was difficult, this idea seemed to have no practical significance.In addition, the emergence of molecular cloning technology provides a way to clone and amplify genes, so Khorana's idea has been forgotten.
On a Friday night in April 1983, when he was driving to his country villa, the idea of "polymerase chain reaction" suddenly flashed out.
In December 1983, Mullis saw the first PCR fragment with a length of 49 bp after 10 cycles by isotope labeling;
In 1985, Kary Mullis invented PCR while working in Cetus.Mullis wants to synthesize DNA primers for sequencing, but he is often worried about not having enough template DNA.
The patent of PCR was applied for on October 25, 1985 and approved on July 28, 1987 (patent No. 4683202). Mullis was the first inventor;
On December 20, 1985, he published the first academic paper on PCR in Science magazine, and Mullis is the co author;
In May 1986, Mullis made a special report in Cold Spring Harbor Laboratory, and the world began to learn PCR methods.[1]
PCR Principle
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DNASemi reserved replicationIt is an important way for biological evolution and generation.Double stranded DNA can be denatured by a variety of enzymesUnrotationSingle strand, with the participation of DNA polymerase, according toBase complementary pairing principleCopy into the same two molecular copies.It was found in the experiment that DNAhigh temperatureDenaturation and chain breaking can also occur when the temperature decreasesRenaturationbecomeDouble chain。Therefore, the denaturation and renaturation of DNA are controlled by temperature change, and the design is addedprimerDNA polymerase and dNTP can replicate specific genes in vitro.
However, DNA polymerase will be inactivated at high temperature. Therefore, new DNA polymerase must be added to each cycle, which is not only cumbersome but also expensive, restricting the application and development of PCR technology.
The discovery of thermostable DNA polymerase Taq enzyme has a milestone significance for the application of PCR. The enzyme can withstand high temperatures above 90 ℃ without inactivation, and does not need to add enzymes in each cycle, which makes PCR technology very simple, and also greatly reduces the cost. PCR technology has been widely used and gradually applied to clinical practice.
The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on the oligonucleotide primers complementary to both ends of the target sequence.PCR consists of three basic reaction steps of denaturation annealing extension: ① denaturation of template DNA: after heating the template DNA to about 93 ℃ for a certain timeDouble chainOr the double stranded DNA formed by PCR amplification is dissociated to form a single strand so that it can be combined with the primer to prepare for the next round of reaction; ②Annealing of template DNA and primers（Renaturation）: After the template DNA is heated and denatured into a single strand, the temperature drops to about 55 ℃Complementary sequencePaired combination; ③Primer extension: DNA template primer combination at 72 ℃DNA Polymerase(such as TaqDNA polymerase), using dNTP as reaction material, target sequence as templatecomplementary base pairing With the principle of semi reserved replication, a new semi reserved replication chain complementary to the template DNA chain is synthesized, and more "semi reserved replication chains" can be obtained by repeating the three processes of cyclic denaturation annealing extension, and this new chain can become the template for the next cycle.It takes 2-4 minutes to complete a cycle, and 2-3 hours to expandTarget geneThe amplification is several million times larger.[3]
Reaction preparation
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PCR reaction system
10 × amplification buffer
10μl
4 dNTP mixtures (final concentration)
100~250 μ mol/L for each
Primer (final concentration)
5 ～ 20 μ mol/L for each
Template DNA
0.1～2μg
Taq DNA Polymerase
5～10 U
Mg2+(final concentration)
1～3mmol/L
Supplement double distilled water
100 μl
DNTP, primer, template DNA, Taq DNA polymerase and Mg2+The dosage (or concentration) of can be adjusted according to the experiment, and the above table only provides approximate reference values.
Five elements of PCR reaction: There are five main substances participating in the PCR reaction, namely: primer (PCR primer is a DNA fragment, and the primer for DNA replication in cells is a RNA chain), enzyme, dNTP, template and buffer (Mg is required2+）。
There are many primer design methods, which are determined by the purpose of PCR in the experiment, but the basic principles are the same.The enzymes used in PCR mainly come from two sources: Taq and Pfu, which are respectively from two different thermophages.Taq amplification is efficient but easy to occurMismatch。Pfu amplification efficiency is weak but has error correction function.Therefore, different choices must be made according to the actual needs.
The template is DNA for amplification, which can be any source, but there are two principles: the first is that the purity must be high, and the second is that the concentration cannot be too high to avoid inhibition.
The composition of buffer solution is the most complex, which generally includes four effective components except water: buffer system, which generally uses HEPES or MOPS buffer system;Monovalent cation, generally potassium ion, but under special circumstances, ammonium ion can also be used;The divalent cation, namely magnesium ion, is determined according to the reaction system and does not need to be adjusted except for special circumstances;Auxiliary ingredients, such as DMSO and glycerin, are mainly used to maintain enzyme activity and help unwind DNA.[3]
PCR Primer Design
There are two primers in the PCR reaction, namely 5 'end primer and 3' primer.The primer design is based on a single strand of DNA (usually information strand), and the 5 'end primer is the same as a small segment of DNA sequence on the 5' end of the fragment to be amplified;The 3 'end primer complements a small segment of DNA sequence located at the 3' end of the fragment to be amplified.
(1) Basic principles of primer design
Primer length: 15-30bp, usually about 20bp.
Primer base: The content of G+C should be 40-60%. Too little G+C has poor amplification effect, and too much G+C is easy to produce non-specific bands.ATGC is the bestrandom distribution, avoid more than 5purineorPyrimidine nucleotideA string of arranged references for.
There should be no complementary sequence 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 nonspecific amplification region should not exceed 70%. The 8 consecutive bases at the 3 'end of the primer should not have complete complementary sequences outside the region to be amplified, otherwise it is easy to lead to nonspecific amplification.
The base at the 3 'end of primer, especially the last and penultimate base, should be paired strictly. The best choice is G and C.
The 5 ′ end of the primer can be modified.For example, add restriction enzyme sites, introduce mutation sites, label with biotin, fluorescent substances, digoxin, and add other short sequences, includingInitial codon、Termination codonEtc.
The basic requirement of sample processing is to remove impurities and partially purify nucleic acid in the sample.Most samples need to go through SDS andproteaseK processing.Bacteria that are difficult to break, availablelysozymeAdd EDTA for treatment.The crude DNA was extracted and purified with phenol and chloroform, and then precipitated with ethanol as a template for PCR reaction.
Control of reaction
The buffer solution of PCR reaction provides appropriate pH and some ions
The total concentration of magnesium ion should be higher than that of dNTPs, usually 1.5mmol/L
Substrate concentration dNTP is prepared with equimolar concentration, 20~200umol/L
TaqDNA polymerase 2.5U (100ul)
The primer concentration is generally 0.1~0.5umol/L
Reaction temperature and cycle times
Denaturation temperature and time 95 ℃, 30s
Annealing temperature and time are about 5 ℃ lower than the Tm value of primer, generally 45~55 ℃
Extension temperature and time 72 ℃, 1min/kb (within 10kb)
Tm value=4 (G+C)+2 (A+T)
Cycle times: generally 25~30 times.The number of cycles determines the yield of PCR amplification.When the initial concentration of template is low, the number of cycles can be increased to achieve effective amplification.However, the number of cycles can not be increased indefinitely.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 products no longer increases with the increase of the number of cycles. The so-called "plateau period" appears.[4]
Cycle parameters
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Predenaturation
Complete denaturation of template DNA and complete activation of PCR enzyme are crucial to the success of PCR. It is recommended to refer to the reagent manual for heating time. Generally, the activation time of unmodified Taq enzyme is two minutes.[5]
Denaturation step
In general, 95 ℃ and 30 seconds are enough to completely denature various target DNA sequences during circulation, and the time of this step can be shortened if possible.If the denaturation time is too long, the enzyme activity will be damaged, and if the target sequence is too short, the denaturation will not be complete, which will easily lead to amplification failure.[5]
primer annealing
The annealing temperature needs to be determined from many aspects. Generally, it is based on the Tm value of the primer as a reference, and it is properly reduced according to the length of amplification as the annealing temperature.Then make predictions on the basis of this experiment.Annealing temperature has great influence on the specificity of PCR.[5]
Primer extension
The primer extension was generally carried out at 72 ℃ (the optimal temperature for Taq enzyme).But when the amplification length is short andannealing temperature When it is high, this step can omit that the extension time depends on the length of the amplified fragment. It is generally recommended to be more than 1000 bp, and the derivative containing Pfu and its derivatives is set at 1 min/kbp.[5]
Number of cycles
Most PCR contain 25-35 cycles, and too much PCR is easy to produce non-specific amplification.[5]
Final extension
After the last cycle, the reaction was maintained at 72 ℃ for 10-30 min. The primer was extended completely and the single chain product was annealed into double chains.[5]
step
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The standard PCR process is divided into three steps:
DNA denaturation: (90 ℃ - 96 ℃):Double chainDNA template is heated,hydrogen bondBreak to form single strand DNA
Annealing: (60 ℃ - 65 ℃): The system temperature decreases, and the primer combines with the DNA template to form a local double strand.
Extension: (70 ℃ - 75 ℃): atTaq enzymeUnder the action of (at about 72 ℃, the activity is the best), dNTP is used as raw material, starting from the 3 'end of the primer and extending from 5' to 3 'end, to synthesize DNA chains complementary to the template.
Each cycle undergoes denaturationannealingAnd extension, the DNA content will be doubled.As shown in the figure, some PCR can be replicated in a short time even if the Taq enzyme activity is not optimal because the amplification region is very short. Therefore, the two-step method can be changed, that is, annealing and extension are conducted at 60 ℃ - 65 ℃ at the same time, so as to reduce a temperature rise and fall process and improve the reaction speed.[5]
testing
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The PCR reaction amplified a high copy number, and the next step of detection became the key.fluorescein（Ethidium bromide，EB）Staininggel electrophoresisIt is the most commonly used detection method.The specificity of electrophoresis is not very high, so the primer dimer is nonspecificHybridIt is easy to cause misjudgment.But because of its simplicity, it has become a mainstream detection method.In recent yearsfluorescent probeAs a representative detection method, there is a trend to gradually replace electrophoresis.[5]
Reaction characteristics
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Strong specificity
The specificity of PCR reaction is determined by:
① Specific and correct combination of primer and template DNA;
② Base pairing principle;
③ The fidelity of Taq DNA polymerase synthesis reaction;
④ The specificity and conservatism of target genes.
The correct combination of primer and template is the key.The combination of primer and template and the extension of primer chain followBase pairingPrincipled.The fidelity of the polymerase synthesis reaction and the high temperature resistance of TaqDNA polymerase make the template and primer combine in the reaction（Renaturation）Can be carried out at a higher temperature, combinedSpecificitySignificantly increased, amplified targetsgene segmentWhich can be kept highAccuracy。By selecting target gene regions with high specificity and conservatism, the degree of specificity will be higher.
High sensitivity
The amount of PCR products is increased exponentially, which canPikeThe initial template to be tested at the order of (pg=10-12) was amplified to the level of microgram (μ g=- 6).One target cell can be detected from one million cells;In virus detection, the sensitivity of PCR can reach 3 RFUs（Plaque forming unit）；staybacteriologyThe minimum detection rate was 3 bacteria.[1]
Simple and fast
High temperature resistant Taq DNA polymerase is used for PCR reaction. After the reaction solution is added at one time, the denaturation annealing extension reaction is carried out on the DNA amplification solution and water bath, and the amplification reaction is generally completed in 2-4 hours.Generally used for amplification productsElectrophoretic analysis, not necessaryisotopeNo radioactive pollution, easy to promote.
Low purity requirements
It is not necessary to isolate virus or bacteria and culture cells, and crude DNA products and RNA can be used as amplification templates.It can be directly used for clinical samples such as bloodCoelomic fluidDNA amplification test of washing solution, hair, cells, living tissues, etc.[1]
common problem
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The electrophoresis detection time of PCR products is generally within 48 hours, and some of them are best detected on the same day. After more than 48 hours, the bands are irregular or even disappear.
false negative
No amplification bands were found.The key links of PCR reaction include ① preparation of template nucleic acid, ② quality and specificity of primer, ③ quality of enzyme and use of ethidium bromide, ④ PCR cycle conditions.To find out the reasons, we should also analyze and study the above links.
Templates: ① templates contain miscellaneous proteins, ② templates contain Taq enzyme inhibitors, ③ templates contain proteins that have not been digested completely, especially histones in chromosomes, ④ templates lose too much or inhale phenols when extracting and preparing templates. ⑤The template nucleic acid was not completely denatured.When the quality of the enzyme and primer is good, there is no amplification band, which is most likely due to the digestion of the sample. There is something wrong with the template nucleic acid extraction process, so it is necessary to prepare an effective and stable digestion solution, and the program should also be fixed and should not be changed at will.[1]
negative
Note that sometimes you forget to add Taq enzyme or ethidium bromide.Primer: The quality of the primer, the concentration of the primer, and whether the concentration of the two primers is symmetrical are the common reasons for PCR failure, unsatisfactory amplification bands, and easy dispersion.SomeBatch NoOfPrimer synthesisThere is a problem with the quality. One of the two primers has a high concentration and the other has a low concentration, resulting in inefficient asymmetric amplification. The countermeasures are: ① select a good primer synthesis unit. ②The primer concentration depends not only on the OD value, but also on the primer stock solutionagarose gel electrophoresis, there must be a primer band, and the brightness of the two primer bands should be roughly the same. If one primer has a band, and one primer has no band, PCR may fail at this time, andPrimer synthesisThe unit shall negotiate for settlement.If one primer has high brightness and the other has low brightness, balance its concentration when diluting the primer. ③Primers should be stored in small quantities with high concentration to prevent deterioration of primers due to repeated freeze-thaw or long-term refrigeration
Degradation failure. ④primer design Unreasonable, if the primer length is not enough, it will form between primersDimerEtc.
Mg2+Concentration: Mg2+Ion concentration has a great impact on PCR amplification efficiency. If the concentration is too high, the specificity of PCR amplification will be reduced. If the concentration is too low, the PCR amplification yield will be affected and even the PCR amplification will fail without amplification bands.
Change of reaction volume: usually the volume used for PCR amplification is 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 detection. When making a small volume, such as 20ul, and then making a large volume, you must model 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, which are very likely to cause false negative;If the annealing temperature is too low, it can cause non-specific amplification and reduce specific amplification efficiency. If the annealing temperature is too high, it will affect the combination of primer and template and reduce PCR amplification efficiency.Sometimes it is necessary to use a standard thermometer to check the denaturation, annealing and extension temperature in the amplification instrument or water dissolving pot, which is also one of the reasons for PCR failure.
Target sequence variation: if the target sequence mutation or deletion affects the specific binding of primer and template, or the deletion of a segment of the target sequence causes the loss of complementary sequence between primer and template, the PCR amplification will not be successful.[1]
false positive
The PCR amplification bands appeared were consistent with the target sequence bands, sometimes the bands were more regular and brighter.
Inappropriate primer design: the selected amplification sequence has homology with the non targeted amplification sequence, so the PCR product amplified during PCR amplification is a non targeted sequence.If the target sequence is too short or the primer is too short, false positives are likely to occur.Primers need to be redesigned.
Cross contamination of target sequences or amplification products: There are two reasons for this contamination: one is cross contamination of the whole genome or large segments, which leads to false positives.This false positive can be solved by the following methods: Be careful and gentle during operation to prevent the target sequence from being sucked into the sampling gun or splashed out of the centrifuge tube.Except for enzymes and materials that cannot withstand high temperature, all reagents or equipment shall be sterilized under high pressure.All centrifuge tubes and sample injection gun heads used shall be disposable.If necessary, reaction tubes and reagents shall be irradiated with ultraviolet light before adding samples to destroy existing nucleic acids.The second is the pollution of small pieces of nucleic acid in the air. These small pieces are shorter than the target sequence, but there are certainHomology。They can be spliced with each other, and after complementing with primers, PCR products can be amplified, resulting in false positives, which can be alleviated or eliminated by nested PCR.
Non specific amplification band appears:
The band after PCR amplification is inconsistent with the expected size, or large or small, or there are specific amplification bands and non-specific amplification bands at the same time.The reasons for the emergence of non-specific bands are: first, the primer and target sequence are not completely complementary, or the primer polymerization is formedDimer。The second is Mg2+High ion concentrationannealing temperature Too low, and too many PCR cycles.The second is the quality and quantity of the enzyme. Some enzymes from some sources are prone to have non-specific bands while the enzyme from another source does not. When the enzyme quantity is too high, non-specific amplification may occur.The countermeasures are as follows: redesign primers when necessary.Reduce the amount of enzyme or replace the enzyme from another source.Reduce the amount of primers, properly increase the amount of templates, and reduce the number of cycles.Properly increase the annealing temperature or use the two temperature point method (93 ℃ denaturation, 65 ℃ annealing and extension).
Flaky drag or smear:
Sometimes smear bands, sheet bands or carpet like bands appear in PCR amplification.The reason is often due to excessive enzyme amount or poor enzyme quality, high dNTP concentration2+It is caused by too high concentration, too low annealing temperature and too many cycles.The countermeasures are: reduce the amount of enzyme, or replace the enzyme from another source. ②Reduce the concentration of dNTP.Properly reduce Mg2+Concentration.Increase the amount of templates and reduce the number of cycles.[3]
clinical application
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It is used to treat infectious diseases, tumors and genetic diseases.[6]
Infectious diseases
The most valuable application of PCR in medical laboratory science is the diagnosis of infectious diseases.In theory, as long as there is a pathogen in the sample, PCR can detect it.General laboratories can also detect 10~100 copies of genes, while current pathogen antigen detection methods generally require 105-7 pathogens to detect.The detection of pathogens by PCR solves the "window period" problem of immunological detection, which can determine whether the disease is in a recessive or subclinical state.
Quantitative PCR research data have shown that the number of pathogens is related to the severity, infectivity and treatment effect of infectious diseases.Many studies have shown that after HIV infection, the length of incubation period and the severity of clinical symptoms are significantly related to the amount of virus in the blood;Some studies have also shown that when the HIV viral load is lower than a certain value, there is no infectivity.
In the quantitative study of hepatitis B virus and hepatitis C virus, it was found that the number of viruses was related to the efficacy of some drugs.For example, interferon therapy is not sensitive to those with high copies of hepatitis virus, but sensitive to those with low copies;Some drugs can significantly reduce the high copy of the virus.[6]
tumour
Oncogene expression increases and mutations in manytumourEarly and benign stages can occur.PCR technology can not only effectively detect gene mutations, but also accurately detect the expression of oncogenes, which can be used for early diagnosis, typing, staging and prognosis of tumors.
Almost all patients with chronic myeloid leukemia can detect the formation of BCR/ABL fusion gene caused by proto oncogene translocation. Quantitative PCR technology can determine the number of trace residual malignant cells by detecting the expression of BCR/ABL fusion gene, which can be used as a basis for therapeutic effect and for estimating the risk of recurrence.
someVirusesThe carcinogenic effect is also related to the viral load. The FQ-PCR test results of EB viral load have been used for early detection and follow-up of nasopharyngeal carcinoma.[6]
Hereditary disease
The first clinical application of PCR technology is to detect sickle cells andβ - thalassemiaThe gene mutation of.Mutation and deletion of genes will cause imbalance in the expression of various globins. FQ-PCR is used to detect the difference in the expression of various globin genesThalassemiaEffective means of diagnosis.[6]
Test contamination
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The greatest feature of PCR reaction is that it has great amplification ability and high sensitivity, but the headache problem is that it is easy to be polluted. A very small amount of pollution can cause false positives.[6]
Causes of pollution
1. Cross contamination between specimens: the contamination of specimens mainly includes the contamination of the container for collecting specimens, or the cross contamination between specimens caused by the loose sealing of the container when the specimens are placed, or the specimens stuck outside the container;During the extraction process of sample nucleic acid template, the sample room is polluted due to the sample aspirating gun pollution;Some microbial samples, especially viruses, can diffuse with or form aerosols, resulting in mutual pollution.
2. Contamination of PCR reagent: It is mainly due to the contamination of sampling gun, container, double distilled water and other solutions by PCR nucleic acid template during the preparation of PCR reagent.
3. Contamination of PCR amplification products: This is the most important and common contamination problem in PCR reaction.Because the copy amount of PCR product is large (generally 1013 copies/ml), which is far 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 ignore and most likely to cause PCR product pollution is aerosol pollution.Aerosols can be formed when the air and liquid surfaces rub, and the reaction tube is shaken violently during operation. Aerosols can be formed and polluted when the cap is opened, when the sample is aspirated, and when the sample is aspirated repeatedly by the contaminated injection gun.It is calculated that an aerosol particle can contain 48000 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 that use cloned plasmids as positive controls.Because the content of cloned plasmid in unit volume is quite high, in addition, more tools and reagents are needed in the purification process, and the plasmid in living cells has strong vitality due to the simplicity of growth and reproduction of living cells.The possibility of pollution is also great.[6]
Pollution monitoring
A good laboratory should always pay attention to pollution monitoring, consider whether there is pollution caused by what reasons, so as to take measures to prevent and eliminate pollution.
1. Positive control: PCR positive control should be set up in the establishment of PCR reaction laboratories and general inspection units. It is an important reference mark for the success of PCR reaction and whether the position and size of product bands meet the theoretical requirements.The positive control should select the sample with medium amplification and good repeatability, which is identified as the product by various tests. If the recombinant plasmid is used as the positive control, the content should be low rather than high (less than 100 copies).However, the positive control, especially the recombinant plasmid and high concentration of positive samples, may pollute the detection or amplification samples.Therefore, when a certain PCR reagent is stable after its own use and the tester knows it well, the positive control can be dispensed with in future experiments.
2. Negative control: negative control must be made every PCR experiment.It includes:
(1) Sample control: If the tested sample is serum, use the identified normal serum as the control;If the sample to be examined is a histiocyte, use the corresponding histiocyte as a control.
(2) Reagent control: PCR amplification is performed without adding template DNA or RNA to the PCR reagent to monitor whether the reagent is contaminated.
3. Repeatability test.
4. Primers from different regions were selected for PCR amplification.[6]
