1 Fundamentals and concepts of polymerase chain reaction
1.1 Basic principles
The replication of DNA in cells is a complex process. The basic factors involved in replication are: DNA polymerase, DNA ligase, DNA template, RNA primers synthesized by initiating enzymes, nucleotide materials, inorganic ions, suitable pH, and several enzymes and protein factors that unwind the superhelix and double helix of DNA.
Polymerase chain reaction is a DNA replication reaction in a test tube, the basic principle is similar to the in vivo, the difference is that the heat-resistant Taq enzyme instead of DNA polymerase, with synthetic DNA primers instead of RNA primers, heating (denaturation), cooling (annealing, insulation (extension) and other methods to change the temperature of the DNA can be replicated, repeated denaturation, annealing, extension cycle, you can make DNA infinite amplification. The specific process of polymerase chain reaction is as follows.
The polymerase chain reaction system is warmed to about 95°C, and the double-stranded DNA template unravels into two single strands, a process known as denaturation. The temperature is then lowered below the Km value of the primer, and the 3′ and 5′ ends of the primer each bind to the complementary regions of the two single-stranded DNA templates, a process known as annealing. When the temperature of the reaction system is raised to about 70°C, the heat-resistant Taq DNA polymerase catalyzes the sequential addition of four deoxyribonucleotides to the 3′ ends of the primers in a complementary manner to the nucleotide sequence of the template DNA, forming a nascent DNA strand. Each cycle approximately doubles the number of DNA molecules in the reaction system. Theoretically, it increases to 2^n times by cycling several times. When after 30 cycles, the DNA yield reaches 2^30 copies, which is about 10^9 copies. The polymerase chain reaction amplification process is shown in Figure 8-1. Since in practice the amplification efficiency does not reach 2-fold, it should therefore be (1+R)^n, with R being the amplification efficiency.
1.2 Factors involved in the polymerase chain reaction reaction system and their roles
The factors involved in the polymerase chain reaction reaction mainly include template nucleic acid, primers, TaqDNA polymerase, buffer, Mg2+, deoxynucleotide triphosphate (dNTP), reaction temperature and cycle number, and polymerase chain reaction instrument. Their roles are described as follows.
(I) Template nucleic acid
The template nucleic acid used for the polymerase chain reaction can be either DNA or RNA. when RNA is used as the template, reverse transcription is first performed to generate cDNA, and then the normal polymerase chain reaction cycle is carried out. Nucleic acid templates come from a wide range of sources and can be extracted from cultured cells, bacteria, viruses, tissues, pathological specimens, archaeological specimens, etc.
The amount of DNA template added to the polymerase chain reaction reaction is typically 100-100000 copies, and the current state of the art has made it possible to prepare the corresponding cDNA library from a single cell. the right amount of DNA template can reduce the base mismatch caused by multiple cycles of the polymerase chain reaction.
Usually a linear DNA molecule is used for the template DNA, in case of a cyclic plasmid, it is better to cut it into a linear molecule with enzymes first, because cyclic DNA is compounded too quickly.
(II) Primers
Primers determine the specificity and length of polymerase chain reaction amplification products. Therefore, primer design determines the success or failure of the polymerase chain reaction reaction.
There are two types of primers in the polymerase chain reaction, namely, 5′ end primer, which is an oligonucleotide with the same sequence as the 5′ end of the template, and 3′ end primer, which is an oligonucleotide complementary to the 3′ end of the template. The basic requirements for the primers are: ① The short length of the primer will affect the specificity of the polymerase chain reaction, requiring 16-30bp, because 4^16=4.29×10^9, which is already larger than 3×10^9bp of the mammalian genome to ensure specific binding; too long primers make the extension temperature exceed the optimal temperature of Taq DNA polymerase (74 degrees), which will also affect the specificity of the product. The specificity of the product will also be affected. ②The content of G+C is generally 40%-60%. ③The four bases should be randomly distributed, and there should not be more than three consecutive identical purines or pyrimidines present. Especially at the 3′ end of the primer, there should not be 3 consecutive G or c, otherwise it will make the primer mis-complement the G or c enriched region of the nucleic acid, and affect the specificity of the polymerase chain reaction. The primers themselves should not have complementary sequences that cause their own folding, at least the primers themselves should not have more than 3bp consecutive complementary bases. ⑤ The two primers should not be complementary to each other, especially their 3′ ends. A pair of primers should not have more than four consecutive bases complementary to each other to avoid creating primer dimer. ④ The homology between primers and non-specific target regions should not exceed 70% or have 8 consecutive complementary bases homologous, otherwise it leads to non-specific amplification. ①The 3′ end of the primer is the point where the extension is triggered. Therefore it should not be mismatched. As ATCG causes mismatch with certain pattern, the A at the 3′ end of the primer has the greatest impact, therefore, try to avoid the first base at the 3′ end of the primer is A. The 3′ end of the primer should not be the third base of the encoding codon either, so as not to affect the amplification specificity because of the simplicity of the codon 3rd position. The 5′ end of the primer can be modified, including the addition of an enzyme cleavage site, labeling with biotin, fluorescent substances, digoxin, etc., introduction of mutation sites, introduction of promoter sequences, introduction of protein-binding DNA sequences, etc. The design of the primers is best guided by computer software.
The concentration of primers in the reaction system is generally required to be between O.1 and 0.5 μmol. Too high concentrations tend to generate primer dimers, or non-specific products.
The primer Tm value is related to the annealing temperature and is calculated as Tm=4(G+C)+2(A+T). The primer Tm value is preferably in the range of 55-80°C, with close to 72°C being the best.
(C) Heat-resistant TaqDNA polymerase
Chien isolated heat-stable polymerase in 1976, and Erlich isolated and purified Tq heat-stable polymerase suitable for polymerase chain reaction in 1986, which laid the foundation for polymerase chain reaction to become a practical technology, and is now produced by recombinant genes. Taq DNA polymerase has a molecular weight of 94 kD, and the enzyme ratio at 75°C is 150 bs/molecule. The reaction temperature is too high or too low to affect its elongation, Taq lei has a high thermal stability. Experiments showed that the half-lives were 40 min, 30 min and 5 min at 92.5°C, 95°C and 97.5°C, respectively.
The purified Taqase has no 3′-5′ exonuclease activity in vitro, thus no corrective reading function, and can cause mismatches during amplification. The number of mismatched bases is influenced by temperature, Mg2+ concentration and number of cycles. Typically, the mismatch rate of 30 cycles of Taqase is about 0.25%, which is higher than the mismatch rate of Klenow enzyme. taqase produces a shift mutation rate of 1/30,000 and a base substitution rate of 1/8000 in each cycle. applying low concentration of dNTP (20 μmol/L each), 1.5 mmol/L Mg2+ concentration, and a temperature higher than 55°C for replication can improve the fidelity of Taqase, when the average mismatch rate is only 5×10^-6 times/(nucleotide* cycle).
Taqase has a TdT-like activity similar to that of end-transferase and can add – a base to the 3′ end of the newly generated double-stranded product, especially dATP being the easiest to add. Therefore, in order to clone the polymerase chain reaction product into the vector, two treatments can be used: one is to construct the dT-vector; the other is to use Klenow enzyme to remove the A from the 3′ end, that is, after the polymerase chain reaction reaction, first inactivate the Taqase by heating at 99℃ for 10min, adjust the Mg2+ concentration to 5-10mmol/L, add 1-2U Klenow fragment, and act for 15-20min at room temperature. After 15-20 min of action, the A at the 3′ end is cut off and the Taqase also has reverse transcription activity. The Taqase also has reverse transcription activity. Similar activity to reverse transcriptase occurs at 68°C at 2-3 mmol/L Mg2+ concentration. The reverse transcription activity is better in the presence of Mg2+, and using this activity, it can be directly used in RNA-polymerase chain reaction, especially for amplification of short fragments.
In the past, polymerase chain reaction amplification with Taq enzyme, generally can only amplify DNA fragments less than 400bp, after the modification of the structure and function of the enzyme, as well as the improvement of polymerase chain reaction methodology, now can amplify DNA in more than 20kb.
The amount of Taq enzyme added in the polymerase chain reaction is also very important, too little of course is not good, too much on the one hand waste, but also lead to non-specific amplification, usually 1-2.5U Taq enzyme per lOOμl reaction solution is good. It is best to determine the optimal enzyme concentration in the range of 0.5-5U. Another issue is that although Taqase is a tool enzyme with good thermal stability, care should be taken to store it at -20°C.
(iv) Buffer
Buffers provide the appropriate pH for polymerase chain reaction reactions with certain ions, commonly 10-50 mmol/L. Tris-HCI (pH 8.3-8.8) buffer. The buffer containing 50 mmol/L KCl facilitates the annealing of primers. Some people and add calf serum albumin (100 μg/L) or gelatin (0.0%) or Twen20 (0.05%-0.1%) or dithiothreitol (DDT, 5 mmol/L), etc., which are thought to protect Taq enzyme.
(E) Mg2+Taqase activity requires Mg2+. too low Mg2+ concentration, Taqase activity significantly reduced; Mg2+ concentration is too high, and the enzyme catalyzes non-specific amplification. mg2+ concentration also affects the annealing of the primer, the temperature of the unchaining of the template and polymerase chain reaction products, the generation of primer dimer, etc. Taqase activity is only related to the concentration of free Mg2+, while the polymerase chain reaction reaction system, all phosphate groups in dNTP, primers, DNA templates, etc. can bind to Mg2+ and reduce the free concentration of Mg2+. Therefore, the total amount of Mg2+ should be 0.2-0.25 mmol/L higher than the concentration of dNTP. If the reaction system contains chelating agents such as EDTA, a part of Mg2+ can also be bound off.
To obtain the optimal concentration of Mg2+, the following optimization method can be used. First, no Mg2+ is added to the polymerase chain reaction buffer small, and a certain amount from the configured 10 mmol/L Mg2+ storage solution is added to each reaction tube, starting with a concentration gradient of 0.5 mmol/L (0.5,1.0,1.5,2.0,2.5,……5.0 mmol/ L), the approximate concentration range of Mg2+ could be determined from the electrophoresis results after the polymerase chain reaction reaction, and then the optimum concentration of Mg2+ could be precisely determined by increasing and decreasing several concentrations above and below this concentration by 0.2 mmol/L.
(vi) dNTP
dNTP is the raw material for the synthesis of polymerase chain reaction reactions. The concentration of each dNTP should be equal, and the usual concentration range is 20-200gmol/L. Within this range, the balance between the amount of polymerase chain reaction products, specificity and fidelity of the reaction is optimal. For example, when each dNTP is 20μmol/L, 2.6μg of 400bp DNA can be theoretically produced. keeping the concentrations of the four dNTPs above their Km values (10-15μmol/L) maintains the fidelity of base incorporation; if the concentration of dNTP is greater than 50mmol/L, the Taqase activity can be inhibited.
(VII) Reaction temperature and number of cycles
1. Denaturation temperature and time
The denaturation step in the polymerase chain reaction is very important. If the template DNA and the polymerase chain reaction product cannot be denatured completely, the polymerase chain reaction cannot be successful. The denaturation temperature and time too high will affect the Taqase activity, the usual denaturation temperature and time are 95 ℃, 30 s, sometimes with 97 ℃, 15 s. Although the DNA strand in the denaturation temperature when the two strands separated only a few seconds, but the reaction tube inside to reach the required temperature still need a certain amount of time, the group to extend the time appropriately. To ensure that the template DNA can be thoroughly denatured. Preferably 7-10 min, then set the denaturation step to 95°C/min in subsequent cycles.
When amplifying 100-300bp short fragments, it is also advisable to use a rapid two-step polymerase chain reaction method, i.e. denaturation (94-97°C), annealing and extension (55-75°C).
To prevent evaporation of the reaction solution at denaturation temperature, 1-2 drops of liquid paraffin can be added in the reaction tube.
2. Denaturation temperature and time
The denaturation temperature determines the specificity of the polymerase chain reaction, and the appropriate denaturation temperature should be 5°C below the primer Tm value. The annealing temperature is too low, causing non-specific amplification; increasing the annealing temperature can improve the specificity of amplification, so the annealing temperature should be strictly specified. The annealing reaction time is generally 1 min.
At the beginning of the first cycle of the polymerase chain reaction, the reaction starts at a temperature much lower than the Tm value, and since Taq enzyme is still active at low temperatures, it is possible that non-specific products or primer dimers may appear due to non-specific ligand pairs of primers and templates, and then the non-specific products are amplified repeatedly throughout the polymerase chain reaction later on, making the polymerase chain reaction a serious failure. To try to eliminate this non-specific amplification, a hot start can be used. There are several methods of hot start: one method is to add an anti-Taqase antibody to the polymerase chain reaction system. The antibody binds to the Taqase and causes inhibition of Taqase activity. Therefore, at the beginning, although the temperature is low and the primer can mismatch with the template, it does not cause non-specific amplification because Taqase is inactive; when thermal denaturation is performed, the antibody is inactivated at high temperature and Taqase is released, it can function to perform specific amplification in the later extension steps
specific DNA polymerization reactions at later extension steps. Another hot-start method uses paraffin to separate the Taqase from the polymerase chain reaction system, so there is no non-specific amplification at room temperature at first either. When warmed to the thermal denaturation temperature, the paraffin melts and the Taqase is mixed with the polymerase chain reaction reverse system, thus functioning in later steps. The use of hot start improves the specificity of polymerase chain reaction amplification.
3. Extension temperature and time
Extension temperature is generally about 72 ℃, at this time Taqase activity of 35-100 nucleotides per second doping, 2kb fragment with 1min has been sufficient, if the DNA fragment is long, amplification time can be extended appropriately. Extension time is too long and can cause non-specific amplification.
4. The number of cycles
The number of cycles depends mainly on the concentration of the initial target molecule, for example, in the initial target molecule for 3×10^5, 1.5×10^4, 1×10^3 and 50 copy number, the number of cycles can be 25-30, 30-35, 35-40 from 40-45 respectively. too many cycles will increase the amount of non-specific products and the number of base mismatches. The increase in amplification products does not become exponential in an exponential manner late in the polymerase chain reaction reaction, called plateau effect. The plateau effect may be related to the following factors: lower dNTP and primer concentrations, relatively lower enzyme-to-template ratio, lower enzyme activity after multiple cycles, and incomplete denaturation with higher product concentrations that affect primer extension.
(H) Polymerase chain reaction instrument
There are a variety of imported and domestic polymerase chain reaction instrument. The temperature rise and fall of the instrument can be gas heating, water heating and electric heating block heating. Temperature, cycle number and time parameters are now mostly controlled by computer. The instrument can be selected according to the needs.
Since the polymerase chain reaction method is highly sensitive, a small number of target molecules can be expanded to an infinite number. Therefore, it is important to prevent the polymerase chain reaction amplification products from contaminating the environment and causing subsequent false positives in the polymerase chain reaction. For this reason, the polymerase chain reaction instrument and polymerase chain reaction product detection processes should be separated from specimen preparation and polymerase chain reaction tube preparation as much as possible in space, preferably in separate rooms. The experimental space can usually be divided into specimen processing area, reaction mix preparation and polymerase chain reaction amplification area, product analysis area, etc.
2 PCR examination
PCR is a method for rapid amplification of specific genes or DNA sequences in vitro, so it is also known as the in vitro amplification of genes. The polymerase chain reaction technique is similar to the natural replication process of DNA and its specificity depends on oligonucleotide primers that are complementary to both ends of the target sequence.
2.1 PCR normal values
The body has a normal variety and proportion of flora and the body is in a dynamic and balanced state of health.
2.2 Clinical significance of PCR
Polymerase chain reaction can rapidly and specifically amplify any known target gene or DNA fragment and can easily amplify target genes in starting DNA mixtures at the picogram (pg) level to specific DNA fragments at the nanogram, microgram, and milligram levels. As a result, polymerase chain reaction technology has been rapidly and widely used in various fields of molecular biology since its inception.
Abnormal results: Abnormalities due to various diseases, such as syphilis. (1) Stage I syphilis. The incubation period is 2 to 4 weeks, with a hard dark red mass and shallow ulcers in the external genital area, with cartilage-like hardness and enlarged surrounding lymph nodes. (2) Stage II syphilis. In the first stage of syphilis 1 to 2 months later, the skin and mucous membranes of the body occur symmetrical generalized rash, rash, papules, pustules, etc.. Mucosal spots and flat warts may occur on mucous membranes, which are highly contagious. (3) Stage III syphilis. It can also involve bones, joints, heart and blood vessels, manifesting as aortitis, aortic valve atresia and aortic aneurysm, etc. It invades the nerves as spinal consumption and general paralysis (paralytic dementia), etc.
People who need screening: patients suspected of a specific disease for molecular specific screening.