In a typical PCR reaction system, appropriate buffer, trace template DNA, 4×dNTPs, thermostable polymerase, Mg2 and two synthetic DNA primers should be added. The template DNA was denatured at 94℃ for 65438 0 minutes, the primer and template were annealed at 40 ~ 60℃ for 65438 0 minutes, and extended at 72℃ for 2 minutes. Before the first cycle, the template is pre-denatured for 3-5 minutes; After the last cycle, the sample still needs to be extended for more than 3 ~ 5 minutes to ensure that the amplified DNA is double-stranded DNA. In order to understand the composition, dosage and reaction conditions of each component in PCR reaction, so that people can change them one by one according to different research objects and find the best reaction conditions, the typical reaction conditions provided by Gene Amp DNA Kit of Perkin Elmer Company in Cetus are listed for reference. For the standard buffer used for PCR, see the example of PCR operation. At 72℃, the pH value of the reaction system will drop 1 unit, approaching 7.2. The existence of divalent cations is very important, which affects the specificity and yield of PCR. Experiments show that Mg2 is superior to Mn2, and Ca2 has no effect.
The optimal concentration of1.mg2m2 is 1.5mmol/L (when the concentration of various dNTP is 200mmol/L), but it is not optimal for any combination of templates and primers. When the target sequence is combined with the primer for the first time, the concentration of Mg2 should be adjusted to the best, and the range of concentration change is1~10 mmol/L. Excessive Mg2 is easy to generate non-specific amplification products, while insufficient Mg2 is easy to reduce the yield.
A higher concentration of chelating agent such as EDTA or a higher concentration of negatively charged ionic groups such as phosphate in the sample will combine with Mg2 and reduce its effective concentration. Therefore, DNA used as template should be dissolved in10mmol/l tris-HCl (pH 7.6) 0.1mmol/l EDTA.
DNTP contains phosphate, and the change of its concentration will affect the effective concentration of Mg2. The total concentration of 4×dTNPs in the standard reaction system is 0.8mmol/L, which is lower than that of Mg21.5 mmol/L. Therefore, under the condition of high concentration of DNA and dNTP, the concentration of Mg2 must be adjusted accordingly.
2.Tris -HCl buffer 10 ~ 50mmol/l tris-HCl buffer is used for PCR, but other buffers are rarely used. Tris buffer is a bipolar ion buffer with a pKa of 8.3 (20℃) and a PKA of 0.02 1/℃. Therefore, when the pH of 20 mmol/L Tris is 8.3 (20℃), the real pH is between 7.8 and 6.8 under typical thermal cycling conditions.
3.KCl concentration of 50 mmol/L K concentration can promote primer annealing. However, the research shows that when the concentration of NaCl is 50 mol/L, when the concentration of KCl is higher than 50 mol/L, the activity of Taq enzyme will be inhibited, and little or no KCl has little effect on the PCR results.
4. Gelatin Gelatin and BSA or nonionic detergent can stabilize the enzyme. The general dosage is 100μg/ml, but the current research shows that good PCR results can be obtained with or without addition, with little effect.
5. Dimethyl sulfoxide (DMSO) is useful in PCR using Klenow fragment; Adding 10% DM-SO is beneficial to reduce the secondary structure of DNA, which makes the template with high content of (G C)% easy to be completely denatured. It is easier to directly sequence PCR products when DMSO is added to the reaction system, but when it exceeds 10%, the activity of Taq DNA polymerase will be inhibited. Therefore, DMSO is not used in most cases. In the PCR reaction system, the final concentration of dNTP higher than 50 mol/L will inhibit the activity of Taq enzyme, and the use of low concentration of dNTP can reduce the wrong incorporation of nucleotides at the start and extension of non-target sites, while high concentration of dNTP will easily lead to wrong incorporation, and too low concentration will inevitably reduce the yield of reactants. The usual concentration of PCR is 50 ~ 200μ mol/L, which should not be lower than10 ~15 μ mol/L. The concentration of four dNTP should be the same, and any one of them will induce the wrong incorporation of polymerase, slow down the synthesis speed and terminate the reaction prematurely.
The factors that determine the minimum concentration of dNTP are the length and composition of the target sequence DNA. For example, in the reaction system of 100μl, if the concentration of 4×dNTPs is 20μmol/L, basically 2.6μg DNA or 400bp sequence of 10pmol can be synthesized. 50μmol/L 4×dNTPs can synthesize 6.6μgDNA, while 200μmol/L is enough to synthesize 25μg/DNA.
Generally, the pH value of dNTP solution purchased from manufacturers is not adjusted. Adjust the pH value of dNTP storage solution to 7.0 with 1mol/l NaOH to ensure that the pH value of the reaction is not lower than 7. 1. Free nucleotide freeze-dried powder purchased in the market should be neutralized with NaOH after being dissolved, and then quantified by ultraviolet spectrophotometer. In a typical PCR reaction mixture, the enzyme concentration is 2.5U/μl, and the commonly used range is1~ 4U/100μ L. Due to different DNA templates and primers and other conditions, the amount of polymerase is different, and too much enzyme will lead to the increase of non-specific products.
Due to the different formulations, production conditions and activity definitions used by manufacturers, the performance of TaqDNA polymerase supplied by different manufacturers is also different.
Cetus Company defines enzyme as: 1 enzyme unit refers to the enzyme needed to mix 10 mmol dNTP into acid-insoluble components at 74℃ for 30min under the following analytical conditions. The determination time was 10min, which was converted into a dose of 30min.
The analytical conditions were 25nmol/L TAPS (sodium trimethylolpropane sulfonate, pH9.3.25℃), 50 μm ol/L KCl, 2 mmol/l MgCl2.1mmol/l β-me (mercaptoethanol), 200 mmol/L dATP, dTTP and dGTP, 6540 mmol/l. Single-stranded and double-stranded DNA or RNA can be used as PCR samples. If the starting material is RNA, the first cDNA must be obtained by reverse transcription. Although PCR can only use a very small number of samples, even the DNA of a single cell, in order to ensure the specificity of the reaction, ng-level cloned DNA, μg-level single-copy chromosome DNA or 104 copy of the fragment to be amplified are also used as starting materials. The template can be crude, but it cannot be mixed with any protease, nuclease, Taq DNA polymerase inhibitor and protein that can bind to DNA.
The size of DNA is not the key factor, but when using extremely high molecular weight DNA (such as genomic DNA), such as ultrasonic treatment or digestion with rare restriction enzymes (such as Sal 1 and Not 1), the amplification effect is better. The amplification efficiency of closed-loop target DNA is slightly lower than that of linear DNA, so it is best to linearize the plasmid first when it is used as a reaction template.
The concentration of template target sequence varies from case to case, which is usually beyond the control of the experimenter. In the experiment, a set of control reactions can be set according to the inverse reduction of the known target sequence (1ng, 0. 1ng, 0.00 1ng, etc.). ) to test whether the sensitivity of the amplification reaction meets the requirements. Agarose gel electrophoresis is often used in practical work. Generally, 1% ethidium bromide (EB) is added to the electrophoresis buffer or gel (every 100ml 100μl), and then the prepared 1% ~ 2% agarose gel (prepared with electrophoresis buffer) is put into the electrophoresis tank and put into the sample to be tested. The concentration of agarose should be selected according to the size of the separated DNA fragment, which is generally 1.5% ~ 2%, and the electrophoresis voltage is 75 V. When the sample is within 65438±0cm from the gel end, cut off the power supply, take out the gel, and directly observe the results under the ultraviolet lamp.
Because ethidium bromide can form a conjugate with double-stranded DNA, it can emit fluorescence under ultraviolet lamp, which enhances the fluorescence intensity of EB by 80 ~ 100 times, so the gel after electrophoresis can be directly observed under ultraviolet lamp. Generally, the amount of DNA observed by naked eyes can reach 10ng, and its fluorescence intensity is directly proportional to the DNA content.
The swimming speed of DNA molecules in gel is determined by charge effect and molecular effect. The former is determined by net charge, while the latter is related to molecular size and configuration. According to the size of DNA molecules, the gel concentration can be adjusted differently. Conditional laboratories can also use polyacrylamide gel electrophoresis (PAGE) to analyze the amplified DNA fragments. PCR technology must have reasonable synthetic primers and extracted sample DNA, then carry out automatic thermal cycling, and finally identify and analyze the products. At present, the design and synthesis of primers can only be carried out in a few research institutes with strong technical strength, and clinical application can only be carried out by purchasing PCR detection kits. There are many factors affecting the automatic thermal cycle of PCR. For different DNA samples, the addition amount of various components in PCR reaction and the temperature cycle parameters are inconsistent. Several main influencing factors are introduced as follows.
First, the temperature cycle parameters.
In the automatic thermal cycle of PCR, the most critical factors are denaturation and annealing temperature. As the operation example shows, the conditions of denaturation, annealing and extension are: 94℃60s, 37℃60s, 72℃ 120s, * * * 25 ~ 30 cycles, and the amplified fragment is 500bp. Here, the time of each step should be calculated after the reaction mixture reaches the required temperature. In the automatic thermal cycler, it takes 30 ~ 60s to change the original temperature of the mixed solution to the required temperature. The length of the lag time depends on several factors, including the type of reaction tube, wall thickness, volume of mixed solution, heat source (water bath or heating block) and temperature difference between the two steps. We should pay full attention to and consider when setting the thermal cycle, and measure each instrument.
Another important consideration about thermal cycle time is the distance between two primers; The longer the distance, the longer it takes to synthesize the full-length target sequence. The reaction time given above is based on the most suitable target sequence with a synthetic length of 500bp. The following describes the choice of various temperatures.
1. template denaturation temperature denaturation temperature is the temperature that determines the melting of double-stranded DNA in PCR reaction. If the denaturation temperature is not reached, the single-stranded DNA template will not be generated and PCR will not be started. If the denaturation temperature is low, the denaturation will be incomplete, and the DNA double-stranded will renaturate quickly, thus reducing the yield. Generally take 90 ~ 95℃. Once the sample reaches this temperature, it should be quickly cooled to the annealing temperature. DNA denaturation takes only a few seconds, not too long; On the contrary, the time at high temperature should be shortened as much as possible to maintain the activity of Taq DNA polymerase, and the highest denaturation temperature after adding Taq DNA polymerase should not exceed 95℃.
2. primer annealing temperature annealing temperature determines PCR specificity and yield; High temperature specificity is strong, but if it is too high, the primer can not be firmly combined with the template, and the DNA amplification efficiency decreases; Low temperature leads to high yield, but too low temperature will cause mismatch between primers and templates and increase non-specific products. Usually, starting from the reaction condition of 37℃, a series of controlled reactions are set to determine the optimal annealing temperature for a specific reaction. It can also be inferred from the (G C)% content of the primer to grasp the starting point of the test. Generally, the annealing temperature is 5℃ lower than the melting temperature TTm of the amplification primer, which can be calculated according to the formula:
Ta = Tm -5℃= 4(G C) 2(A T)-5℃
Where a, t, g and c respectively represent the number of corresponding bases. For example, for a primer with 20 bases, if the content of (G. C)% is 50%, the starting point of Ta can be set at 55℃. At the typical primer concentration (such as 0.2μmol/L), the annealing reaction can be completed in a few seconds, without long-term annealing.
3. The selection of primer extension temperature depends on the optimum temperature of Taq DNA polymerase. The general temperature is 70 ~ 75℃. At 72℃, the standard rate of enzyme-catalyzed nucleotides can reach 35 ~ 100 nucleotides/second. The length of 1kb can be extended per minute, and its speed depends on the composition of buffer, pH value, salt concentration and the nature of DNA template. If the amplified fragment is shorter than 150bp, the extension step can be omitted and become a two-temperature cycle, because Taq DNA polymerase can complete the synthesis of short sequences at annealing temperature. For short sequence fragments between100 and 300 BP, it is effective to adopt fast and simple two-temperature cycle. At this time, the primer extension temperature is the same as the annealing temperature. For DNA fragments above 1kb, the extension time can be controlled at 1 ~ 7min according to the fragment length. At the same time, gelatin or BSA reagent should be added to PCR buffer to keep Taq DNA polymerase active and stable for a long time. 1.5% ~ 20% glycerol is helpful to amplify DNA fragments of about 2.5kb or longer.
4. The cycle number of conventional PCR is generally 25 ~ 40 cycles. The common mistakes are too many cycles, serious background nonspecific and increased complexity. Of course, if the number of cyclic reactions is too small, the yield will be low. Therefore, under the premise of ensuring the product yield, try to reduce the number of cycles.
After amplification, the sample was cooled and stored at 4℃.
Second, primer primer design
In order to amplify the template DNA, two oligonucleotide primers should be designed first. Primers are actually two oligonucleotide fragments complementary to the target DNA sequence to be amplified. The distance between the two primers determines the length of the amplified fragment, and the 5' ends of the two primers determine the two 5' end positions of the amplified product. It can be seen that primers are the key to determine the length, position and result of PCR amplification fragments, and primer design is more important.
The necessary condition of primer design is that the target DNA sequence complementary to the primer must be known, and the sequence between two primers may be unclear. These two known sequences are generally 15 ~ 20 bases, and two primers corresponding to them can be synthesized by DNA synthesizer. In addition, the principles usually followed in primer design include:
1. Primer length According to statistical calculation, the probability that an oligonucleotide sequence with a length of about 17 bases appears in the human genome is 1 time. Therefore, the length of primers is generally not less than 16 nucleotides, but not more than 30 nucleotides, and the optimal length is 20-24 nucleotides. This short oligonucleotide will not form a stable hybrid at the polymerization temperature (72℃). Sometimes a sequence complementary to the template can be added to the 5' end, such as restriction sites or promoters, to complete gene cloning and other special needs; Biotin labeling or fluorescence labeling at the 5' end of primers can be used for various purposes, such as microbial detection. Sometimes the reason why the primer does not work is unknown, which can be solved by moving the position.
2. The primer with (G C)% content should be uniform in composition, and try to avoid containing the same basic polymer. The content of (G C)% in the two primers should be as close as possible, and when the content of (G C)% in the amplified fragment is known, it should be close to the fragment to be amplified, generally 40% ~ 60% is better.
3. The obvious secondary structure, especially hairpin structure, should be avoided in primers. For example:
4. There should be no complementarity between the two primers, especially at the 3' end of the primer. Even if it is inevitable, the complementary bases at the 3' end should not exceed 2 bases, otherwise "primer dimer" or "primer dimer" will be easily generated. Primer dimer is essentially a double-stranded DNA fragment with a length similar to that of two primers formed by extension of one primer on another primer sequence under the action of DNA polymerase, which is a common by-product of PCR, and sometimes even the main product.
In addition, there is no homologous sequence between the two primers, especially more than six oligonucleotide fragments have the same base, otherwise the two primers will compete with each other for the same site of the template; Similarly, primers and other sequences of target DNA or sample DNA to be amplified cannot have homologous sequences of more than 6 bases. Otherwise, the primers will combine with other sites, reducing specific amplification and increasing non-specific amplification.
5. Pairing DNA polymerase at the 3' end of primer means adding a single nucleotide at the 3' end of primer, so the matching requirements of 5 ~ 6 bases at the 3' end of primer and target DNA must be accurate and strict to ensure the effective amplification of PCR.
Whether the PRIMER design is reasonable or not can be verified by computer retrieval with PCRDESN software and American Primer software.
The synthesized oligonucleotide is preferably purified by chromatography or PAGE to remove impurities, such as short chains that have not been synthesized in full length. The purified primers were stored in 25% acetonitrile solution at 4℃, which could inhibit the growth of microorganisms. In general, the unused primer should be stored in the refrigerator at -20℃, and the primer can be stored in liquid for 6 months, and can be stored for 1 ~ 2 years after freeze-drying.
Third, DNA polymerase
1956, kornberg and others discovered DNA polymerase from the extract of Escherichia coli, and obtained the pure product of DNA polymerase I, which is composed of polypeptide chains with molecular weight of 109000 and can be decomposed into two fragments by subtilisin. One fragment has polymerase activity, the molecular weight is 76,000, and the exonuclease activity of nucleic acid is 3'→5, that is, Klenow fragment. The other fragment has a molecular weight of 34,000 and has 5' →' 3' nucleic acid exonuclease activity. So DNA polymerase has several functions: one is polymerization, which uses DNA as a template to add deoxynucleotides in dNTP to the 3-OH terminal one by one. Second, it has the exonuclease activity of' 3' → 5' nucleic acid, and can identify and eliminate mismatched primer ends, which is related to the correction function during replication. The third is the exonuclease activity of 5'→3' nucleic acid, which can hydrolyze nucleotides from the 5' end and remove mismatched nucleotides through the action of several nucleotides. 1985, Muhlis and others invented the PCR method. After completing the PCR of β -globin with Klenow fragment, many laboratories in the world consider using heat-resistant DNA polymerase instead of Klenow fragment for PCR, which makes the research of heat-resistant polymerase develop rapidly. Thermostable DNA polymerases have been isolated and purified from many bacteria living at 60℃ (Bacillus stearothermophilus) to 87℃ (Streptococcus solvates), but some enzymes cannot tolerate the temperature required for DNA denaturation, so they cannot be used for PCR.
1.Taq DNA polymerase replacing Klenow fragment of Escherichia coli DNA polymerase ⅰ with Taq DNA polymerase is the key to make PCR widely used. Klenow fragment can not tolerate the denaturation temperature of double-stranded DNA at 95℃, so new enzymes should be added every cycle; Taq DNA polymerase can withstand the high temperature of 93 ~ 95℃, avoiding the complicated operation of adding polymerase, increasing the annealing and extension temperature, reducing the interference of non-specific products and DNA secondary structure on PCR, and improving the specificity, yield and sensitivity of PCR. The main differences are as follows: ① The optimum temperature of Klenow enzyme is 37℃, and the amplified products are not all the target sequences, so it needs to be detected with probes. Taq enzyme has high yield and strong specificity. The optimum temperature is 74 ~ 75℃. Therefore, the annealing temperature can be increased, the annealing rigor can be improved, and the extension of mismatched primers can be reduced. ② In the later period of the cycle, the amount of enzyme is gradually insufficient, resulting in a gentle slope. For Klenow enzyme (both starting with 1μg genomic DNA) and Taq enzyme, the number of cycles reaching the level wave is 20 and 30 respectively. The extension length of Taq enzyme is less than 10kb, and that of Klenow enzyme is less than 400bp.
Taq enzyme was isolated from thermophilic bacterium YT 1. The strain was isolated by Brock from hot springs in Yellowstone National Park on 1969. As a standard strain of thermophilic bacteria, its growth temperature is 70 ~ 75℃. DNA polymerase with molecular weight of 60 ~ 68 kDa and specific activity of 2000 ~ 8000 U/mg was isolated. Later, Kary Mullis of Cetus Company isolated the pure enzyme with specific activity of 200,000 U/mg and molecular weight of 93,965,438+00. The optimum temperature of this 9.4KDa enzyme is 75 ~ 80℃, and the binding rate (Kcat) with simple nucleotides can reach 150 nucleotides (nt)/s enzyme molecule. With M 13 as template, it was extended with 30bp primers rich in g.c., at 70℃, KACT >: 60nt/s; 24 nt/s at 55℃; It is 65438 0.5 nt/s at 37℃ and as low as 0.25nt/s at 22℃. When the temperature is higher than 90℃, the DNA synthesis activity is very poor. Under this high temperature condition, primers and templates can not be firmly combined.
In the mixed solution of PCR reaction, the time for Taq enzyme to maintain 50% activity at 92.5℃, 95℃ and 97.5℃ was 65438 0 30, 40 and 5 ~ 6 min respectively, and the highest temperature in the tube was 95℃ in 50 PCR cycles. Fashion can maintain 65% vitality, 20s per cycle. The half-life of Taq enzyme is 40 minutes at 95℃, so the denaturation temperature selected in PCR cycle should not be higher than 95℃.
Taq enzyme can be produced by gene recombination, and its trade name is Ampli Taq (Cetus Company). The complete gene of Taq enzyme is 2499bp long, expressed and produced in Escherichia coli, and contains 832 amino acids. The amino acid sequence has 38% identity with Escherichia coli DNA polymerase ⅰ, including its binding to dNTP. There are primer and template action regions in Taq enzyme.
Taq enzyme has a 5' →' 3' nucleic acid exonuclease activity that depends on DNA synthesis, so there is an annealed 3'- phosphorylated "blocker" on the template, which will be cut off one by one without preventing the extension from the upstream primer chain. However, for the synthetic oligonucleotide primers labeled with 5'-32P, no degradation was found in either single strand or template renaturation, so this activity will not affect the PCR results. Taq enzyme has no exonuclease activity of 3' →' 5' nucleic acid, and if dNTP is mixed incorrectly, this enzyme has no correction ability. Therefore, when Taq enzyme is used for PCR, there are many midpoint mutations in the product, which is not conducive to cloning. Generally, the doping ratio is1.25×10-4 ~10-5 (4× dntps concentration is 200μmol/L, Mg2 is 1.5mmol/L, and it is annealed at 55℃). However, the lack of exonuclease activity of 3'→5' nucleic acid is beneficial to sequencing.
2. Factors affecting enzyme activity The activity of Taq enzyme is influenced by Mg2+. When herring sperm DNA was used as template, the total dNTP concentration was 0.7 ~ 0.8 mmol/L and Mg2 concentration was 2.0mmol/L, the activation ability was the highest. When the concentration exceeds this value, it will produce inhibition. The inhibitory activity of 10mmol/l magnesium chloride reached 40% ~ 50%. DNTP can bind with Mg2, so the free Mg2 is only the remaining amount after binding. When the total dNTP concentration is as high as 4 ~ 6 mmol/L, the activity of Taq enzyme will decrease by 20 ~ 30%, that is, substrate inhibition.
When the concentration of dNTP is low, the yield and specificity of PCR increase, which is suitable for labeling biotin and radioactive elements by amplification incorporation method. When 100μl PCR solution contains 40μmol/L dNTP, it is enough to synthesize 2.6μg DNA(dNTP consumes half).
Based on the incorporation of dNTP in herring sperm DNA at 70℃ 10 min, the standard condition is 100%.
The pure 9.4KDa Taq enzyme does not contain the nucleic acid exonuclease activity of 3'→5' nucleic acid. The error classification rate depends on the concentration of dNTP. But Taq enzyme has the DNA-dependent nucleic acid exonuclease activity of 5'→3' nucleic acid. There is almost no degradation ability for single-stranded oligonucleotide labeled with 5 ′→ 3 ′ 32p or hybridization with MB template.
The medium concentration of KCl can increase the activity of Taq enzyme synthesis by 50% ~ 60%, and the optimum concentration of KCl is 50 mol/L, while the higher concentration can inhibit it. >; 200mmol/L KCl can inactivate the enzyme.
Adding 50mmol/L NH4Cl or NH4Ac or NaCl can produce moderate inhibition or no effect.
Low concentrations of urea, DMSO, DMF or formamide have little effect, but Tween 20/NP40 can eliminate the inhibitory effect of SDS(0.0 1% and 0. 1%).
3. stoffel fragment of the second generation thermostable DNA polymerase: Stoffel of Cetus Company removed the 5'→3' nucleic acid exonuclease active fragment (289 amino acids at the N-terminal) of TaqDNA polymerase, which is called Stoffel fragment. The half-life of Taq DNA polymerase at 97.5℃ is increased from 5 ~ 6 min to 20min, and the enzyme fragment is also more conducive to the amplification reaction of two or more template sites, that is, multiplex PCR.
VentTMDNA polymerase: It was isolated and purified from Thermococcus litoralis, a thermophilic bacterium that can grow at 98℃ by New England Biological Laboratory Company, so it was named Vent enzyme. Some of its enzymatic properties are better than Taq DNA polymerase. It can tolerate the high temperature of 100℃ and keep its activity for more than 2 hours. It has the ability to correct the exonuclease activity of 3'→5' nucleic acid, and the false amplification probability is twice as low as that of Taq DNA polymerase. Later, the company transplanted the deep nozzle DNA polymerase gene into the GB-D strain isolated from the nozzle of a deep-water submarine (20 10m). The strain can grow at 104℃, and the half-life of deep nozzle DNA polymerase reaches 23h at 95℃ (nozzle enzyme 6.7h, Taq enzyme 1h).
4.rTth reverse transcriptase RT-PCR is developing rapidly at present, so the research on heat-resistant RNA-dependent DNA polymerase has also made progress. Some experiments show that Taq DNA polymerase has RNA-dependent DNA polymerase activity, but its activity is weak. In 199 1, Cetus company introduced a RTTH retrovirus, which has good RNA-dependent and DNA-dependent thermostable DNA polymerase activities, and the two activities depend on Mn2M2 respectively, so that the enzyme activities can be controlled separately. Using this enzyme, only 250ng of total RNA is needed to carry out RT-PCR effectively, and specific DNA fragments can be obtained, which is very beneficial to the development of reverse transcription PCR.
The research of thermostable DNA polymerase has made great progress and played an important role in the development of PCR. It is believed that with further research, people's understanding and application of heat-resistant DNA polymerase will further develop.
The research of PCR has developed rapidly in China, and its key reagent, thermostable DNA polymerase, has been isolated and purified in several laboratories such as Institute of Genetics of Fudan University, Huamei Company and Institute of Basic Medicine of China Academy of Medical Sciences. The latter two strains are thermophilic thermophilic bacteria yt- 1. The former was isolated and purified from the thermophilic bacteria we screened. The Institute of Genetics of Fudan University also successfully cloned the gene of this polymerase, and obtained a thermostable F4DNA polymerase, whose enzymatic properties are very close to Taq DNA polymerase, which provided a guarantee for the development of China PCR.
Fourthly, the factors affecting the specificity of PCR.
Through the above content. It can be seen that there are many factors that affect the specificity of PCR. Let's summarize them here for your reference: ① Rigidity of annealing step: increasing annealing temperature can reduce mismatch hybridization and improve specificity. ② Shortening annealing time and extension time can reduce error initiation and error extension. ③ Primer dimerization is the most common by-product, and reducing the concentration of primers and enzymes can also reduce false start-up, especially primer dimerization. ④ Changing the concentration of magnesium chloride (sometimes KCl) can improve the specificity, which may be a direct effect on Taq enzyme. ⑤ If there is a secondary structure in the template, for example, when the fragment to be amplified is easy to form a hairpin structure, 7- denitrogenation -2'- deoxyguanosine -5'- triphosphate (De7GTP) can be added to the 4×dNTPs in the PCR mixture. If the mixture of de7GTP and dGTP is 3:1(150μ mol/l DE7GTP 50μ mol/l DGTP) instead of 200μmol/l dGTP, the formation of nonspecific products can be prevented.
Verb (abbreviation for verb) enlarges flat slope.
The amplification reaction cannot go on indefinitely. After a certain cycle, the fragment to be amplified no longer grows exponentially but gradually enters a gentle slope. The number of cycles entering the flat slope depends on the number of template copies at the beginning and the total amount of DNA synthesized The so-called flat slope is the late stage of batch PCR cycle. When the synthetic product reaches 0.3 ~ 1 pmol, the original exponential growth rate becomes a flat curve due to the accumulation of products. The reasons why PCR enters the flat slope are: the consumption of primers and dNTP, and the inactivation of Taq enzyme, which will not appear in the standard reaction. In addition, there are several possibilities:
The excess of 1. substrate is due to the fact that the amount of DNA synthesis in the reaction solution is more than that of Taq enzyme. When the reaction solution contains 2.5Utaq enzyme in 100μl, when the amount of DNA synthesis reaches 1μg(3nmol deoxynucleotides), it begins to become substrate surplus. Prolonging the extension time or adding Taq enzyme can overcome this problem. But it is not practical, because every time the next cycle is carried out, the extension time will be doubled, and Taq enzyme will be added to keep exponential growth.
2. The competition of non-specific amplification products is closely related to the above situation. At this time, unwanted DNA fragments and needed fragments compete for polymerase at the same time. In order to overcome this situation, it is necessary to improve the specificity of the reaction so that unnecessary fragments cannot be accumulated in large quantities.
3. When annealing, the single chain of the product will self-combine. During annealing, two single-stranded DNA fragments can also be associated with primers, which will also prevent the product from increasing. This phenomenon will occur when the product concentration reaches 10pmol/ 100μl, which cannot be overcome except dilution.
4. The product is denatured under the condition of high concentration, the product is not completely melted, and the final product is blocked (pyrophosphate, double-stranded DNA).
In a word, the conditions of PCR vary from system to system, and there is no uniform optimal condition. Firstly, the general conditional amplification is selected, and then the parameters are slightly changed, so that the optimization can be achieved and excellent specificity and yield can be obtained.