2 English Reference Polymerase Chain Reaction [WS/T 203-2001Common Terms in Blood Transfusion Medicine]
Pcr [ws/t 203-2001common terms in blood transfusion medicine]
3 Definition of Polymerase Chain Reaction (PCR) refers to the detection method of selectively amplifying DNA or RNA fragments in vitro with primers [1]. This method can be used for blood typing, bone marrow transplantation matching and pathogen detection of transfusion-transmitted diseases in the field of blood transfusion [1].
Polymerase chain reaction (PCR) is a molecular biological experimental method for enzymatic synthesis of specific DNA fragments in vitro, which mainly consists of three repeated thermal cycles: high temperature denaturation, low temperature annealing and appropriate temperature extension. That is, the template DNA is denatured into a single strand at high temperature, and two primers are annealed with complementary sequences on the two template DNA strands at an appropriate temperature by DNA polymerase, and then the annealed primers are extended with four deoxynucleotide triphosphates (dNTPs) as substrates under the catalysis of DNA polymerase. Repeatedly, DNA fragments between two known sequences are amplified by geometric multiples. [2]
4. Historical Review of Polymerase Chain Reaction 4. 1 The earliest idea of nucleic acid amplification in vitro was put forward by Khorana and his colleagues in 197 1: "After DNA denaturation, hybridization with suitable primers, extension of primers with DNA polymerase, and continuous attention to this process, tRNA motifs can be cloned". However, it was difficult to sequence and synthesize oligonucleotide primers at that time. At that time (1970), Smith and others discovered DNA restriction endonuclease, which made it possible to clone genes in vitro, so the early ideas of Khorana and others were forgotten.
4.2 Invention of Polymerase Chain Reaction Until 1985, the epoch-making polymerase chain reaction (PCR) was invented by Mullis, the human genetics laboratory of PECetus Company in the United States, which enabled people to realize their dream of infinitely amplifying nucleic acid fragments in vitro. Its principle is similar to DNA replication in vivo, but it provides suitable conditions for DNA synthesis in vitro in test tubes. Firstly, the Klenow fragment of Escherichia coli DNA polymerase was used to amplify a specific fragment in the human genome. Because the enzyme is not heat-resistant, Klenow enzyme should be added again after heating denatured DNA. When operating multiple samples, this process is time-consuming, laborious and error-prone. The application of thermostable DNA polymerase made the PCR reaction easier to automate, and then PECetus Company introduced the first PCR thermal cycler, which made the automation of this technology a reality. Mullis and others won the Nobel Prize in 1993 for this technology.
The development of PCR and related technologies is amazing. The first and second seminars on PCR technology were held in 1988 and 1990 in the United States and Britain respectively. The first session mainly discusses the application of PCR and the optimization of the technology itself; The main topic of the second meeting is the latest progress of human genome project and PCR. This fully reflects biologists' attention to PCR.
(Table) Related Techniques of Polymerase Chain Reaction
The main purpose of the name is to amplify unknown gene fragments by degenerate primer amplification.
Nested PCR improved the sensitivity and specificity of PCR, and analyzed the mutation.
Simultaneous detection of multiple mutations or pathogens by multiplex PCR
The unknown sequences on both sides of the known sequence were amplified by reverse PCR, resulting in product mutation.
Amplification of unknown genomic DNA by PCR with a single specific primer
Amplification of unknown cDNA by unilateral primer PCR with known sequence
Analysis of sequences at different ends by anchored PCR
Synergistic PCR can reduce primer dimer and improve PCR specificity.
Immobilized PCR is beneficial to the separation of products
Removal of pollution impurities or PCR product residues by membrane combined with PCR
Synthetic or mutated protein DNA fragments were generated by expression cassette PCR.
Connection-mediated PCR DNA methylation analysis, mutation and cloning
Amplification of cDNA end by RACEPCR
Quantitative PCR quantifies mRNA or chromosome genes.
In situ PCR was used to study the proportion of cells expressing genes.
Identification of bacterial or genetic effects by presumed PCR
Universal primer PCR amplification of related genes or detection of related pathogens
Messenger amplification phenotype mapping and synchronous analysis of a small number of cell mRNA.
With the development of other amplification technologies, PCR and related technologies, new amplification technologies are constantly being born. These technologies have their own advantages and disadvantages, and complement each other with PCR, and some of them can be combined. Isomorphism has become a big family of nucleic acid amplification technology in vitro. We believe that with the development of molecular biology technology, new members will appear in this family.
Other in vitro nucleic acid amplification techniques (table) use transcription-dependent amplification system (TAS) to detect HIV ligase chain reaction (LCR) to detect point mutation and autonomous sequence replication (3SR) to study RNA. Clinical application, forensic equal strand displacement amplification (SDA) detection, gene identification Qβ replicase system increased the sensitivity of probe detection, and the circulating probe reaction increased the sensitivity of probe detection by 6. 1. (1) Ligase chain reaction (LCR) is a new DNA amplification and detection technology in vitro, which is mainly used for point mutation research and target gene amplification. It was designed and invented by Backman 1997, and it was used to detect point mutation in the target gene sequence and was patented.
The basic principle of LCR is to use DNA ligase. Double-stranded DNA fragments were specifically connected, and the target gene sequence was amplified in large quantities by three-step repeated cycle of denaturing annealing connection.
The amplification efficiency of LCR is similar to that of PCR. Using thermostable ligase as LCR only needs two temperature cycles, denaturation at 94℃min, renaturation and ligation at 65℃, and the cycle is about 30 times. Its product detection is also convenient and sensitive. At present, this method is mainly used in the research and detection of point mutation, the detection of microbial pathogens and directed mutation. It can also be used for the diagnosis of polymorphism and products of single-base genetic diseases, the identification of microbial species, the study of point mutation of oncogenes and so on.
6.2 Nucleic acid sequence-dependent amplification (a) Nucleic acid sequence-based amplification (NASBA), also known as self-sustaining sequence replication (3SR) or regrowth sequence replication technology. Guatelli et al. first reported this technology in 1990.
NASBA is mainly used for RNA amplification, detection and sequencing.
The basic method is as follows: adding primers and samples into the amplification reaction solution, destroying the secondary structure of RNA molecules at 65℃ 1min, cooling to 37℃, adding reverse transcriptase, T7RNA polymerase and RNase H, reacting at 37℃ for 1 ~ 1.5 hours, and staining with agarose gel electrophoresis and ethidium bromide to obtain the product.
NASBA is characterized by simple operation, no need for special instruments and no temperature cycle. The whole reaction process is controlled by three enzymes, with fewer cycles, high fidelity, higher amplification efficiency and better specificity than PCR.
6.3 Transcription-dependent amplification system (a) Transcription-based amplification system (TAS) was reported by Kwen et al. in 1989, which is mainly used to amplify RNA.
The main feature of TAS is its high amplification efficiency, because its RNA copy number increases exponentially with 10. TAS only needs six circulating target sequences, and the copy number can reach 2× 106. Another feature of TAS is its high specificity. Because TAS can only endure six temperature cycles, its miscibility is low and its hybridization with dextran beads is also high.
Although this method has high specificity and sensitivity, its circulation process is complicated, and reverse transcriptase and T7RNA polymerase need to be added repeatedly, which needs further study.
6.4 Qβ replicase reaction (a) Cassin is equal to 1972. The self-replication function of RNA template catalyzed by Qβ replicase is reported for the first time. Its natural template MDV 1RNA can be amplified to 109 at room temperature for 30min. In 1986, Chu et al. reported that target sequence-specific probes with biomarkers can hybridize with avidin-linked MDV 1RNA. After the unbound MDV 1 is eluted, Qβ replicase is added to amplify and replicate the copy of MDV 1, and then the copy is detected by ethidium bromide staining or hybridized with a second probe from the same source.
Qβ replicase is an RNA-directed RNA polymerase with three characteristics: ① RNA synthesis can be started without the guidance of oligonucleotide primers. ② It can specifically recognize the unique RNA folding structure formed by intramolecular base pairing in RNA gene. ③ Inserting a short nucleic acid sequence into the unfolded structure region of the natural template MDV 1 RNA of Q β replicase does not affect the replication of the enzyme. Therefore, if the nucleic acid probe is inserted into this region, its sequence may be amplified by Qβ replicase.
Lizardi et al., 1988, transcription of MDV 1 RNA probe from target gene sequence *** MDV 1 plasmid by T7 RNA polymerase. This RNA probe can hybridize with the target sequence, then wash off the non-hybridized probe, add Qβ replicase to amplify the probe, and the amplified probe can be used as a template for exponential amplification. Use the above two methods to test the product. Now this technology has been developed to sandwich hybridization, molecular switching and target-dependent replication.
Application examples of PCR technology: research: gene cloning; DNA sequencing; Analysis mutation; Gene recombination and fusion; Recognition and regulation of protein-binding DNA sequence; Mapping of transposon insertion sites; Detection of gene modification; Construction of synthetic gene; Constructing clone or expression vector; Detection of gene endonuclease polymorphism
Diagnosis: bacteria (spirochete, mycoplasma, chlamydia, mycobacteria, rickettsia, diphtheria, pathogenic Escherichia coli, Shigella dysenteriae, Aeromonas hydrophila, Clostridium difficile, etc. ); Viruses (HTLV, HIV, HBV, HPV, EV, CMV, EBV, HSV, measles virus, rotavirus and parvovirus B19); Parasites (malaria, etc. ); Human genetic diseases (Leishmaniasis, thalassemia, hemophilia, BMD, DMD, cystic fibrosis, etc. )
Immunology: HLA division; Qualitative analysis of T cell receptor or antibody diversity; Gene mapping of autoimmune diseases; Lymphatic factor quantization
Human genome engineering: generating DNA markers by spreading repetitive sequences; Construction of genetic map (detection of DNA, polymorphism or * * * mapping); Construction of physical atlas; Sequencing, expression profile
Forensic medicine: analysis of crime scene specimens; Chladik
Tumors: pancreatic cancer, colon cancer, lung cancer, thyroid cancer, melanoma, hematological malignancies.
Tissue and population biology: genetic clustering research; Animal protection research; Ecology; Environmental science; Experimental legacy.
Paleontology: Archaeology and Museum Specimen Analysis
Zoology: diagnosis of animal infectious diseases.
Botany: detecting plant pathogens, etc.
8 Basic principles and concepts of PCR 8. 1 Basic principles DNA replication in cells is a complex process. The basic factors involved in replication are: DNA polymerase, DNA ligase, DNA template, RNA primer to start enzyme synthesis, nucleotide raw materials, inorganic ions, proper pH, some enzymes to untie supercoiled and double-stranded DNA structures and protein factor.
PCR is a DNA replication reaction in a test tube. Its basic principle is similar to that in vivo. The difference is that thermostable Taq enzyme replaces DNA polymerase, and the synthesized DNA primer replaces RNA primer. By changing the temperature such as heating (denaturation) and cooling (annealing, heat preservation (extension), DNA can be replicated, and repeated denaturation, annealing and extension cycles can make DNA infinitely amplified. The specific process of PCR is as follows:
When the PCR reaction system is heated to about 95℃, the double-stranded DNA template is unbound into two single strands, which is denaturation. Then, the temperature is lowered below the Km value of the primers, and the primers at the 3' end and the 5' end are bound to the complementary regions of the two single-stranded DNA templates respectively. This process is called annealing. When the temperature of the reaction system rises to about 70℃, the thermostable Taq DNA polymerase catalyzes four deoxynucleotides to be added to the 3' end of the primer in turn according to the complementary mode of the template DNA nucleotide sequence to form a new DNA chain. Each cycle will double the number of DNA molecules in the reaction system. Theoretically, it will increase to 2 n times after several cycles. After 30 cycles, the DNA yield reached 2 30 copies, about 10 9 copies. The PCR amplification process is shown in fig. 8 1. In fact, the amplification efficiency is less than 2 times, so it should be (1+r) n, where r is the amplification efficiency.
8.2 Factors involved in the PCR reaction system and their functions The main factors involved in the PCR reaction are template nucleic acid, primers, TaqDNA polymerase, buffer, Mg2+, deoxynucleotide triphosphate (dNTP), reaction temperature and cycle times, PCR instrument, etc. Their functions are described as follows:
(1) template nucleic acid
The template nucleic acid used for PCR can be DNA or RNA. When RNA is used as a template, reverse transcription is firstly performed to generate cDNA, and then normal PCR cycle is performed. Nucleic acid templates are widely available and can be extracted from cultured cells, bacteria, viruses, tissues, pathological specimens and archaeological specimens.
The amount of DNA template added in PCR reaction is generally 100 100000 copies. At present, the corresponding cDNA library can be prepared from a single cell. Appropriate content of DNA template can reduce the base mismatch caused by repeated cycle PCR.
Generally, linear DNA molecules are used as template DNA. If it is a circular plasmid, it is best to cut it into linear molecules by enzyme first, because the circular DNA renaturates too quickly.
(2) Primers
Primers determine the specificity and length of PCR amplification products. Therefore, primer design determines the success or failure of PCR reaction.
There are two kinds of primers in PCR reaction, namely, 5' primer and 3' primer. The 5' end primer refers to the oligonucleotide with the same sequence as the 5' end of the template, and the 3' end primer refers to the oligonucleotide complementary to the 3' end of the template. The basic requirements for primers are as follows: ① The short primer length will affect the specificity of PCR, and 1630bp is needed, because 4 164.29 x 10 9 is larger than 3x10 9bp of mammalian genome, which ensures the specific binding; If the primer is too long, the extension temperature will exceed the optimum temperature of Taq DNA polymerase (74 degrees), which will also affect the specificity of the product. ② The content of G+C is generally 40% ~ 60%. ③ Four bases should be randomly distributed, and there should be no more than three consecutive purines or pyrimidines. Especially at the 3' end of the primer, there should not be three consecutive G or C, otherwise the primer and the G or C enrichment region of nucleic acid will be wrongly complementary, which will affect the specificity of PCR. ④ Primers themselves cannot have complementary sequences, which will cause self-folding. At least the continuous complementary bases of primers themselves cannot be greater than 3bp. ⑤ The two primers can't complement each other, especially their 3' ends. No more than 4 consecutive bases between a pair of primers should be complementary to avoid primer dimerization. ④ The homology between primers and non-specific target should not exceed 70% or there are 8 continuous complementary bases, otherwise it will lead to non-specific amplification. ① The 3' end of the primer is the point where the primer begins to extend. Therefore, there should be no mismatch. Due to the regularity of mismatch caused by ATCG, primer 3' terminal A has the greatest influence, so the first base at the 3' terminal of primer should be avoided as much as possible. The 3' end of the primer should not be the third base of the coding codon, so as not to affect the amplification specificity due to the degeneracy of codon 3. ⑧ The 5' end of the primer can be modified, including adding restriction sites, biotin, fluorescent substances and digoxin markers, introducing mutation sites, introducing promoter sequences, introducing protein-binding DNA sequences, etc. The design of primers is best guided by computer software.
In the reaction system, the concentration of primers is generally required to be between 0. 10.5 μ mol. If the concentration is too high, it is easy to produce primer dimer or non-specific products.
The Tm value of the primer is related to the annealing temperature, and the calculation formula is Tm4(G+C)+2(A+T). The Tm value of the primer is preferably in the range of 5580℃, preferably close to 72℃.
(3) thermostable TaqDNA polymerase
Chien isolated thermostable polymerase in 1976, and Erlich isolated and purified Tq thermostable polymerase suitable for PCR in 1986, which laid the foundation for PCR to become a practical technology and has been produced by gene recombination. Recently, there are many kinds of polymerases that can be used for PCR: Taq enzyme extracted from aquatic thermophilic bacteria, Taq enzyme obtained from thermophilic seedlings, VENT enzyme isolated from thermophilic cocci in Litoralis and Sac enzyme isolated from acid-hot bath sulfurized split seedlings, among which Taq enzyme is the most widely used. The molecular weight of Taq DNA polymerase is 94kD, and the specific activity at 75℃ is 150bs/ enzyme molecule. Too high or too low reaction temperature will affect its elongation, and TaqDNA buds have high thermal stability. Experiments show that the half-lives are 40 minutes, 30 minutes and 5 minutes at 92.5℃, 95℃ and 97.5℃, respectively.
The purified Taq enzyme has no exonuclease activity of 3'5' nucleic acid in vitro, so it has no reading correction function, which may cause mismatch during amplification. The number of mismatched bases is affected by temperature, Mg2+ concentration and cycle times. Usually, the mismatch rate of Taq enzyme after 30 cycles is about 0.25%, which is higher than that of Klenow enzyme. The frameshift mutation rate of Taq enzyme per cycle is 1/30000, and the base substitution rate is 1/8000. The application of low concentration of dNTP (20 μ mol/L each), Mg2+ concentration of 1.5mmol/L and renaturation temperature above 55℃ can improve the fidelity of Taq enzyme, and the average mismatch rate at this time is only 5x10 6 times/(nucleotide * cycle).
Taq enzyme has similar activity to terminal transferase TdT, which can add a base to the 3' end of the newly generated double-stranded product, especially dATP. Therefore, there are two methods to clone PCR products into vectors: one is to construct dT vectors; Second, Klenow enzyme removes 3'- terminal A, that is, after PCR reaction, the Taq enzyme is inactivated by heating at 99℃ 10min, the Mg2+ concentration is adjusted to 5 10mmol/L, and the 12U Klenow fragment is added to remove 3'- at room temperature 1520min.
Taq enzyme also has reverse transcription activity. When the concentration of Mg2+ was 23mmol/L and the temperature was 68℃, reverse transcriptase-like activity appeared. If Mg2+ exists, the reverse transcription activity is better, which can be directly used for RNAPCR, especially for the amplification of short fragments.
In the past, when Taq enzyme was used for PCR amplification, only DNA fragments less than 400bp could be amplified. After the modification of the structure and function of Taq enzyme and the improvement of PCR methodology, more than 20kb DNA fragments can be amplified.
The amount of Taq enzyme added in PCR reaction is also very important. Too little is not good, too much waste leads to non-specific amplification. Generally, it is better to contain 12.5U Taq enzyme per 100μl reaction solution. The optimum enzyme concentration is determined in the range of 0.55U U. Another problem is that Taq enzyme is a tool enzyme with good thermal stability, but it should be stored at 20℃.
(4) buffer solution
Buffer provides suitable pH and some ions for PCR reaction, usually1050mmol/l .. trishci (pH 8.38.8) buffer. Buffer containing 50mmol/L KCl is beneficial to the annealing of primers. Some people also added calf serum albumin (100μg/L) or gelatin (0.0%) or Tween 20 (0.05% 0. 1%) or dithiothreitol (DDT, 5mmol/L), and thought that these substances could protect Taq enzyme.
(5) The activity of 5)Mg2+Taq enzyme needs Mg2+. When the concentration of Mg2+ was too low in summer, the activity of Taq enzyme decreased obviously. The concentration of Mg2+ is too high, which makes the enzyme catalyze nonspecific amplification. The concentration of Mg2+ also affects the annealing of primers, the melting temperature of templates and PCR products, and the generation of primer dimer. The activity of Taq enzyme is only related to the concentration of free Mg2+, but in the PCR reaction system, all phosphate groups in dNTP, primers and DNA templates can combine with Mg2+, reducing the concentration of free Mg2+. Therefore, the total amount of Mg2+ should be 0.20.25mmol/L higher than the concentration of dNTP. If the reaction system contains chelating agent such as EDTA, a part of Mg2+ can also be combined.
In order to obtain the optimal concentration of Mg2+, the following optimization methods can be used. Firstly, Mg2+ was not added to the PCR buffer, and a certain amount of Mg2+ was added to each reaction tube from the prepared 10 mmol/L storage solution. Initially, the concentration gradient increased by 0.5mmol/L (0.5, 1.0, 1.5, 2.0,.
(6) dNTP
DNTP is the synthetic raw material of PCR reaction. The concentration of each dNTP should be equal, and the usual concentration range is 20200 gmol/L. In this range, the balance between the amount of PCR products, the specificity and loyalty of the reaction is the best. For example, when each dNTP is 20μmol/L, 2.6μg of 400bp DNA can be produced theoretically. Keeping the concentration of tetradntp higher than its Km value (10 15μmol/L) can keep the fidelity of base incorporation. If the concentration of dNTP is more than 50 mol/L, the activity of Taq enzyme will be inhibited.
(7) reaction temperature and cycle time
1. denaturation temperature and time
The denaturation step is very important in PCR reaction. If the template DNA and PCR products cannot be completely denatured, the PCR reaction cannot be successful. The more G+C content in DNA molecules, the higher denaturation temperature is needed. Excessive denaturation temperature and time will affect the activity of Taq enzyme. The usual denaturation temperature and time are 95℃ and 30s respectively, and sometimes 97℃ and 15s are used. Although it takes only a few seconds for DNA strands to separate at denaturation temperature, it takes some time for the inside of the reaction tube to reach the required temperature, so it needs an appropriate extension of time. In order to ensure that the template DNA can be completely denatured, it is best to be 7 10min, and then the number of denaturation steps in the subsequent cycle is set to 95℃/min.
When the short fragment of 100300bp is amplified, a fast two-step PCR method can be used, namely denaturation (9497℃), annealing and extension (5575℃).
In order to prevent the reaction solution from evaporating at denaturation temperature, 65438 02 drops of liquid paraffin can be added to the reaction tube.
2. Renaturation temperature and time
The renaturation temperature determines the specificity of PCR, and the suitable renaturation temperature should be lower than 5℃ of the Tm value of primers. The annealing temperature is too low, resulting in non-specific amplification; Increasing annealing temperature can improve the specificity of amplification, so annealing temperature should be strictly controlled. The annealing reaction time is generally 65438 0 minutes.
At the beginning of the first cycle of PCR, the reaction starts at a temperature far below the Tm value. Because Taq enzyme is still active at low temperature, nonspecific products or primer dimers may appear at the nonspecific interface between primers and templates, and then the nonspecific products are repeatedly amplified in the whole PCR reaction, which leads to serious failure of PCR. In order to eliminate this non-specific amplification as much as possible, we can adopt the method of hot start, and there are several methods of hot start: one method is to add anti-Taq enzyme antibody to PCR system. The combination of antibody and Taq enzyme inhibited the activity of Taq enzyme. So at the beginning, although the temperature is low, the primer can be mismatched with the template, but because Taq enzyme is inactive, it will not cause nonspecific amplification; When thermal denaturation is carried out, the antibody is inactivated at high temperature, releasing Taq enzyme, which can play a role and be characterized in the subsequent extension step.
Heterogeneous DNA polymerization. Another hot start method is to separate Taq enzyme from PCR reaction system with paraffin, so that there is no nonspecific amplification at room temperature at first. When the temperature rises to the thermal denaturation temperature, paraffin melts, and Taq enzyme is mixed with PCR negative system, thus playing a role in the following steps. Using hot start can improve the specificity of PCR amplification.
3. Extension temperature and time
Generally, the extension temperature is about 72℃. At this time, the activity of Taq enzyme is 35 100 nucleotides per second, and 1 minute is enough for a 2kb fragment. If the DNA fragment is long, the amplification time can be extended appropriately. Prolonging the extension time will lead to nonspecific amplification.
4. Number of cycles
The number of cycles mainly depends on the concentration of the initial target molecule. For example, when the initial target molecules are 3x10 5, 1.5x 10 4, 1x 10 3 and 50 copies, the number of cycles can be 2530, 3035 and 3540 respectively. Too many cycles will increase the number of nonspecific products and base mismatches. In the late stage of PCR reaction, the increase of amplified products is not exponential, which is called platform effect. The platform effect may be related to the following factors: the concentration of dNTP and primers decreased, the ratio of enzyme to template decreased relatively, the enzyme activity decreased after many cycles, and the denaturation was incomplete after the product concentration increased, which affected the primer extension.
(8) PCR instrument
There are many imported and domestic PCR instruments. The heating and cooling methods of the instrument can be gas heating, water heating and electric heating block heating. Parameters such as temperature, cycle times and time are now controlled by computer. You can choose the instrument according to your needs.
Because of the high sensitivity of PCR, a small number of target molecules can be amplified to an infinite number. Therefore, it is necessary to prevent PCR amplification products from polluting the environment and causing false positive PCR in the future. Therefore, the process of PCR instrument and PCR product detection should be separated from specimen preparation and PCR reaction tube preparation as far as possible, preferably in different rooms. Generally, the experimental space can be divided into sample processing area, reaction mixed preparation and PCR amplification area, product analysis area and so on.
9 polymerase chain reaction examination polymerase chain reaction is a method of rapid amplification of specific genes or DNA sequences in vitro, so it is also called in vitro gene amplification. 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.
9. 1 normal value of polymerase chain reaction The species and proportion of bacteria in the body are normal, and the human body is in a dynamic balance and healthy state.
9.2 Clinical Significance of Polymerase Chain Reaction Polymerase chain reaction can rapidly and specifically amplify any known target gene or DNA fragment, and can easily start the amplification of target gene in DNA mixture at pg level, reaching specific DNA fragments of nanogram, microgram and milligram. Therefore, PCR technology has been widely used in various fields of molecular biology as soon as it came out.
Abnormal results: abnormalities caused by various diseases, such as syphilis. ① Primary syphilis. That is, chancre, the incubation period is 2 ~ 4 weeks, and dark red lumps, superficial ulcers, chondroid hardness and lymphadenopathy appear in the external genitalia. ② Secondary syphilis. After 0/~ 2 months of primary syphilis/kloc-0, symmetrical generalized rashes, macules, papules and pustules appeared on the skin and mucosa of the whole body. Mucosal plaque and verruca plana can appear, which is highly contagious. ③ Tertiary syphilis. It occurs 2 ~ 3 years or even 10 years after infection, and the skin is gelatinous swelling, which can also involve bones, joints, heart and blood vessels. The main manifestations are arteritis, aortic insufficiency and aortic aneurysm. The invaded nerves include spinal tuberculosis, general paralysis (paralytic dementia) and so on.