PCR (polymerase chain reaction) is used to amplify a small amount of target DNA in vitro for analysis. Reaction system: 1 sample DNA; 2 primer, is a synthetic pair of oligonucleotide strands (about 15-20 nucleotides) for amplification between the double primer and the template DNA complementary region The region; 34 dNTPs; 4Tag DNA polymerase, isolated from a Thermos aquaticus (Thermus aquaticus). The optimum temperature of this enzyme is 75-80 Â° C, but it is not deactivated at 95 Â° C for a short time. 5 suitable buffer system and appropriate amount of Mg2+. Reaction process: 1 denaturation: the reaction solution is placed in a PCR machine to raise the temperature (about 90-95 Â° C) to dissociate the DNA double strand; 2 renaturation: cooling (about 60 Â° C or so) annealing, the primer is combined with the template; 3 extension: the temperature is raised to 70-75 Â° C, the complementary strand of the template single strand is synthesized under the guidance of the primer to form a DNA double-stranded fragment; 4 repeat the above process of "denaturation - renaturation - extension". The long-chain DNA formed in the first few cycles is more, but as the reaction progresses, the long-chain DNA increases in arithmetic progression, and the target DNA sandwiched between the two primers increases exponentially, about 20- After 30 cycles, the amplified DNA was mainly the target DNA.
Reagents and solutions
(1) 10 Ã— PCR buffer
500 mM potassium chloride
100 mM Tris-Cl (pH 8.3 at room temperature)
15 mM magnesium chloride (2) 10 mM deoxynucleotide (dNTPs) solution - contains all four dNTPs (pH 8.0)
(3) Heat-resistant DNA polymerase:
1 Taq DNA polymerase is purified from E. coli expressing the DNA polymerase gene of the cloned thermophilic bacteria. This enzyme has 5'â†’3' DNA polymerase and 5'â†’3' exonuclease activity, but lacks 3'â†’5' exonuclease activity. This enzyme consists of a polypeptide having a molecular weight of approximately 94 kilodaltons. Taq DNA polymerase is thermostable and can be used to synthesize single-stranded templates into DNA using primers at higher temperatures. The amount of enzyme required to catalyze a typical PCR is about 2 units. Excessive amounts of enzymes may result in amplification of non-target sequences.
Taq DNA polymerase lacks a calibration function, and the nucleotide incorporation error rate during PCR can reach 2 Ã— 10-4 nucleotides per cycle, which is about 4 times higher than that of the Klenow fragment of E. coli DNA polymerase I. For a 30-cycle cycle of amplification reactions, this incorporation error rate will result in a total error frequency of 0.25%. This frequency seems to increase with increasing concentration. The incorporation of these errors can occur anywhere in the amplification product, both transversion and conversion (but without long deletions, chimerization or insertion).
Definition of unit: One unit means that 10 nmol of deoxyribonucleotides are incorporated into the acidic precipitated substance within 30 minutes at 74oC. The experimental conditions were: 25 mM TAPS (pH 9.3), 50 mM potassium chloride, 2 mM magnesium chloride, 1 mM DTT, 0.5 mg/ml active salmon sperm DNA, 0.2 mM dATP, dCTP, dGTP, dTTP.
2 rTth DNA polymerase is a mixture of thermostable DNA polymerases in the two strains of Thermus thermophilus (Tth) and Thermococcus litoralis (Tli). This mixed enzyme has in particular the dual activity of 5'â†’3' DNA polymerase and 3'â†’5' exonuclease (corrected). This enzyme increases fidelity and yield of long-chain PCR when used in the recommended amounts.
Conventional PCR amplification of target DNA sequences is generally within 5 kb. The rTth DNA polymerase can be amplified from human genomic DNA to a 19.6 kb Î²-globin gene group and a 42 kb fragment of phage lambda DNA.
(4) Agarose gel (5) 10 Î¼M upstream and downstream primers (6) DNA template (7) Control DNA (DNA containing target sequence)
(8) DNA length standard reference
2. Equipment (1) 0.2 ml PCR amplification tube (2) can be preset to the temperature program of the amplification program (PCR instrument)
Method 1. Basic PCR Method The basic PCR method described below can be used as a general guide and starting point for any PCR amplification. Since various PCR reaction conditions (such as the number of PCR amplifications, temperature, concentration of Taq DNA polymerase, primers, magnesium chloride, and template DNA) vary greatly, various PCR reaction conditions should be adjusted according to the specific conditions.
1. Add the ingredients to the 0.2 ml sterile amplification tube in the following order:
Component volume final concentration
10 Ã— PCR buffer 5 Î¼l 1 Ã—
10 mM dNTP solution (pH 8.0) 1 Î¼l each 0.2 mM
10 Î¼M upstream primer 2.5 Î¼l 0.5 Î¼M
10 Î¼M downstream primer 2.5 Î¼l 0.5 Î¼M
5 units / Î¼l heat-resistant DNA polymerase 0.5 Î¼l 2.5 units
DNA template 1-10 Î¼l 1 pg-1Î¼g
Disinfected distilled water to 50 Î¼l
* We advocate that for multi-tube reaction, the mixture can be prepared in one tube and then dispensed into each tube to reduce the loss of reagents and the inaccuracy of each tube.
2. Mix the mixture in the vial and add 50 Î¼l of mineral oil or silicified oil to cover the mixture in the vial.
3. Cap the tube tightly and centrifuge briefly to allow the PCR mixture to sink to the bottom of the tube.
4. The tubules were incubated for 3 minutes at 94 Â°C in a temperature circulator (PCR instrument) to completely denature the template DNA.
5. Perform 25-35 cycles of PCR amplification as follows:
Denaturation 94oC 30 seconds
Annealing 55oC 30 second extension 72oC 1 minute / 1 kb
The PCR samples were incubated at 6.72oC for 10 minutes and maintained at 4oC. Samples can be stored at -20oC until use.
7. The PCR reaction product was analyzed by agarose gel electrophoresis, and the developed DNA was stained with ethidium bromide, and DNA of a suitable molecular weight standard was used as a reference.
Method 2, Hot Start Method The method of hot start PCR is to add Taq DNA polymerase when the reaction temperature is lowered to 80 Â° C to ensure the synthesis of highly specific PCR products.
1. Add all mixtures of PCR reactions, but without Taq DNA polymerase, as described in the basic PCR method.
2. Mix the mixture in the vial and cover with 50 Î¼l of mineral oil or silicified oil.
3. Cap the tube tightly and centrifuge briefly to allow the PCR mixture to sink to the bottom of the tube.
4. The tubules were incubated for 3 minutes at 94 Â°C in a PCR machine to completely denature the template DNA.
5. After incubation at 94oC, maintain the PCR reaction at 80oC.
6. Add 0.5 Î¼l (2.5 U) of Taq DNA polymerase to each PCR tube, taking care to ensure that the enzyme is added to the PCR mixture below the oil.
7. Denaturation, annealing and extension of 25-35 cycles were continued as described in the basic PCR method.
1. PCR primer design:
Primer design may be the most critical factor in the success of PCR amplification. Poor primer design may result in insufficient amplification products or even amplification failure, because poorly designed primers can lead to non-specific amplification and/or formation of primer dimers, which in turn becomes a competitive product of PCR reactions. Thereby further inhibiting the formation of PCR products.
The following factors must be considered in the design of the primers, the most important of which are primer length, solution temperature (Tm), specificity, primer sequence complementation, G/C content and polypyrimidine (T, C) or polypyrene (A). , G) extension, 3'-end sequence, and the like.
(1) Primer length: Since the specificity of the PCR reaction, annealing temperature and time are at least partially related to the length of the PCR primer, the primer length is a key factor for the success of PCR amplification. Typical PCR primers are 15-30 nucleotides in length.
(2) Dissolution temperature (Tm): The H bond is broken by heating to separate or "dissolve" the double-stranded DNA into single-stranded DNA. The dissolution temperature (Tm) refers to the temperature at which half of the double-stranded DNA becomes single-stranded DNA. Tm can be calculated by the following formula: Tm = 2 (A + T) + 4 (G + C).
It should be noted that the PCR reaction has at least two (one pair) primers, and the Tm values â€‹â€‹of the two oligonucleotide primers should be relatively close and cannot be too different. Because primers with higher Tm values â€‹â€‹can be mismatched at lower reaction temperatures, and primers have lower Tm values, higher reaction temperatures may not work. Therefore, if the Tm values â€‹â€‹of the primers are different, This leads to inefficient PCR amplification and even amplification failure.
(3) Primer sequence complementation: When designing primers, there should be absolutely no pairing of internal primers of more than 3 bases. For example, primers have such self-pairing regions, and "springback", that is, partial double-stranded structure will occur during annealing reaction.
(4) G/C content and polypyrimidine (T, C) or poly (A, G) extension: the primer sequence to be selected should be as random as possible, and the G+C content is average - avoid long A+T And areas rich in G+C. The GC content in the base composition of the primer is generally from 45% to 55%. There should be no more G or more C extensions in the selected primer sequences, as it promotes non-specific annealing, and multiple A and multiple T extensions should also be avoided as they can "breathe" and extend the primer-template complex. The efficiency of amplification. At the same time, polypyrimidine (T, C) and poly (A, G) extension should also be avoided.
(5) 3'-end sequence: To control primer mismatch, the position of the 3' end of the PCR primer should be carefully determined.
In summary, PCR primer design should be emphasized, and careful consideration should be given when designing PCR primers. For a successful PCR, several important factors such as primer length, GC content, and 3' sequence must be optimized. The ideal primer should be a mixture of almost random nucleotides, a GC content of 50%, and a length of about 20 bases, thus enabling a Tm value in the range of 56-62oC. When analyzing the potential primer sites of target genes, no single polymer should be considered, there is no obvious tendency for secondary structure formation, no self-complementation, and no obvious homology with other double-stranded target gene sequences. To avoid boring designs, save time and reduce errors, computer programs can be used to optimize the design, selection, and determination of oligonucleotide primers.
The most commonly used primer design software and website is: http://
2. Template DNA:
The template DNA was lysed in 10 mM Tris-Cl (pH 7.6) containing a low concentration of EDTA (<0.1 mM). The DNA concentrations were: mammalian genome (100 Î¼g/ml), yeast genomic DNA (1 Î¼g/ml), bacterial genomic DNA (0.1 Î¼g/ml), and plasmid DNA (1-5 Î¼g/ml).
3. Prevention of contamination: Since PCR can amplify a single DNA molecule, care should be taken to prevent contamination of the reaction system by trace DNA templates, especially in the case of low concentration of target sequences to be amplified.
(1) If possible, add PCR reagents and carry out the reaction in a laminar flow table equipped with an ultraviolet lamp. The UV lamp should be turned on when no workbench is used. PCR-specific microcentrifuges, disposable gloves, complete pipettes, and other necessities should always be placed in the workbench. Since the pipette of the automatic pipette is a common source of contamination, a positive drain pipette with a disposable tip and piston should be used for dosing and pipetting. Prepare a special set of reagents and solutions dedicated to PCR. All glassware was baked at 150oC for 6 hours. All plastic vessels, buffers, tips and centrifuge tubes must be subjected to high pressure before use.
(2) Once you enter the special place where the PCR reagent is added and start working, you should wear a pair of new gloves and should be replaced.
(3) Prepare a set of reagents for your own use, which is divided into small portions, and it is best to set up a special location in the refrigerator near the workbench to save. These reagents must not be used for other purposes. When preparing these reagents, use new glassware, plastic utensils, and pipettes that have never been exposed to any DNA used in the laboratory. After use, this small portion will be discarded and may not be relocated.
(4) Before the microcentrifuge tube containing the PCR reagent is opened, it should be centrifuged (10 seconds) on a microcentrifuge in a dedicated workbench. This allows liquid to deposit on the bottom of the tube, reducing the chance of contaminating the glove or applicator.
(5) It is best to add template DNA after adding all other reaction components, including mineral oil to prevent evaporation. After adding the template to the microcentrifuge tube, cover the tube and gently hit the tube side with a gloved finger to mix the liquid. Repeat the centrifugation (10 seconds) to separate the aqueous and organic phases.
(6) When adding template DNA to the PCR system, be careful not to form a spray, which may contaminate other reactions. All pipe that is not ready to use should be tightly closed. Gloves should be replaced after taking the template DNA tube.
(7) Each PCR must contain positive and negative controls. A positive control is used to monitor the efficiency of the PCR reaction, while a negative control is used to determine if there is contamination of the DNA target sequence.
(8) A positive control reaction (i.e., PCR involving a small number of appropriate target sequences) should be set whenever possible. Proper dilution of the target sequence DNA should be performed at a different location in the laboratory prior to the experiment to prevent the concentrated solution of the target DNA from being brought to the laboratory for PCR.
(9) A control reaction containing no template DNA but containing all other components of the PCR system must be set up. This control tube must be added after all other PCRs have been prepared.
4. What we have described above is only a basic PCR reaction condition. For each specific PCR reaction, the reaction conditions vary greatly and need to be adjusted according to the situation.
(1) Time: The time described above is suitable for PCR reaction in a 0.2 ml thin-walled tube with a reaction volume of 50 Î¼l. The thermal cycler is Perkin-Elmer 9600 or 9700, Master Cycler (Eppendorf) and PTC 100. (MJ Research). If the PCR equipment and reaction volume change, both time and temperature need to be adjusted.
The time of each step should be calculated from the time the reaction mixture reaches the desired temperature. In general, it takes 30-60 seconds for the original temperature of the mixture to reach the desired temperature. The length of the temperature lag time depends on several factors including the type of reaction tube, the volume of the reaction mixture, the heat source (water bath or heating block), and the temperature difference between two successive steps. It is therefore important to adjust the incubation time to compensate for this lag time.
The further the distance between the two oligonucleotide primers, the longer the time required to synthesize the full length of the target sequence. The reaction times given above were formulated on a 1000 nucleotide target sequence.
(2) Temperature: It is a compromise to select the annealing temperature of the oligonucleotide primer to the DNA template. If the annealing temperature is lowered, the amplification efficiency is improved, but the mismatch between the primer and the template can be significantly increased. When the temperature is increased, the specificity of the amplification reaction increases, but the total reaction efficiency decreases. It is therefore desirable to set up a series of control reactions to determine the optimum annealing temperature for a particular amplification reaction.
(3) Number of cycles: The number of cycles of the amplification reaction depends on the concentration of the target DNA in the reaction mixture. If the single copy gene in the mammalian genome is amplified to be agarose or polyacrylamide gel electrophoresis, at least It takes 25 cycles.
Exponential amplification of target sequences is not an unrestricted process. Under normal reaction conditions, after 25-30 cycles of amplification (i.e., about 106-fold amplification), the amount of Taq DNA polymerase enzymes becomes a factor limiting the progress of the reaction. For further amplification, the resulting DNA sample should be diluted 1000 to 10,000 times and then used as a template for new PCR.
(4) Buffer: When the standard buffer is incubated at 72 Â° C, the pH of the reaction system will drop by more than one unit, resulting in a pH of the buffer close to 7.2. The presence of divalent cations is critical, magnesium ions are superior to manganese ions, and calcium ions are ineffective. The optimum concentration of magnesium ions is quite low (1.5 mM), so it is important that the template DNA prepared should not contain high concentrations of chelating agents, such as EDTA; nor should it contain high concentrations of negatively charged ionic groups. , such as phosphate. Therefore, the DNA used as a template should be dissolved in 10 mM Tris-Cl (pH 7.6), 0.1 mM EDTA (pH 8.0).
In a broader context, this standard buffer is effective for oligonucleotide primers of various templates, and is not optimal for any particular combination of template and primer. Therefore, the conditions listed above should be modified and improved as a starting point. Whenever a new combination of target sequences and primers is used for the first time, or when the concentration of dNTPs or primers changes, the concentration of Mg2+ is particularly optimized. dNTPs are the main source of phosphate in the reaction, and any change in concentration will affect the effective concentration of Mg2+. We recommend setting up a set of reactions in which the concentration of Tris-Cl (10 mM) and potassium chloride (50 mM) is the same for each reaction, while the concentration of magnesium chloride is different (0.05-5 mM, 0.5 mM each time). After the end of the reaction, the amount of each amplification product was compared by agarose gel electrophoresis and ethidium bromide staining.
PCR can be automated in a thermal cycler (i.e., a PCR machine) to standardize the various cycles of the reaction.
5. In general, if PCR is first performed with new template DNA, new primers, or newly prepared thermostable DNA polymerase, amplification will not be as good as expected. The PCR reaction conditions need to be carefully adjusted to inhibit non-specific amplification and/or increase the yield of the target DNA.
6. A successful PCR amplification reaction with an apparently identical length of DNA fragment of the expected size, as determined by DNA sequence analysis, Southern hybridization, or restriction map analysis.
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