Five elements of the PCR reaction: There are five main substances involved in the PCR reaction, namely primers, enzymes, dNTPs, templates, and Mg2+.

Primers: Primers are the key to PCR-specific reactions, and the specificity of the PCR product depends on the extent to which the primer is complementary to the template DNA. In theory, as long as any template DNA sequence is known, the complementary oligonucleotide strands can be used as primers, and the template DNA can be amplified in vitro by PCR.

Primers should be designed to follow the following principles:

1 Primer length: 15-30bp, usually about 20bp.

2 Primer amplification span: 200-500 bp is appropriate, and fragments up to 10 kb can be amplified under specific conditions.

3 primer base: G + C content is 40-60% is appropriate, G + C too little amplification effect is not good, G + C too much is prone to non-specific bands. ATGC is preferably randomly distributed to avoid the string arrangement of more than 5 purine or pyrimidine nucleotides.

4 Avoid secondary structure inside the primer to avoid complementation between the two primers, especially the 3' end, otherwise a primer dimer will be formed, resulting in a non-specific amplification band.

The bases at the 3' end of the 5 primers, especially the last and second to last bases, should be strictly matched to avoid PCR failure due to terminal base mismatch.

The 6 primers may or may be added with a suitable restriction site, and the target sequence to be amplified preferably has a suitable restriction site, which is advantageous for digestion analysis or molecular cloning.

7 Primer specificity: Primers should have no significant homology to other sequences in the nucleic acid sequence database. Primer amount: The concentration of each primer is 0.1~1umol or 10-100pmol. It is better to produce the desired result with the lowest primer amount. The high primer concentration will cause mismatch and non-specific amplification, and increase the formation of primers. The opportunity for the polymer.

Enzymes and their concentrations: There are currently two supply of Taq DNA polymerases, one is a natural enzyme purified from the genus Bacillus, and the other is a genetically engineered enzyme synthesized by Escherichia coli. Catalyzing a typical PCR reaction requires an enzyme amount of 2. 5 U (refer to a total reaction volume of 100 ul). If the concentration is too high, non-specific amplification can be caused. If the concentration is too low, the amount of the synthesized product is reduced.

The quality and concentration of dNTP: the quality and concentration of dNTP are closely related to the efficiency of PCR amplification. The dNTP powder is granular, and loses its biological activity if it is improperly preserved. The dNTP solution is acidic and should be formulated to a high concentration with 1M NaOH or 1M Tris. The pH of the HCL buffer is adjusted to 7.0-7.5, and the small amount is dispensed and stored at -20 °C. Multiple freeze-thaw cycles will degrade dNTP. In the PCR reaction, the dNTP should be 50 ~ 200umol / L, especially pay attention to the concentration of the four dNTPs are equal (equal molar preparation), such as when any one of the concentrations is different from the other (higher or lower), It will cause a mismatch. Too low a concentration will reduce the yield of PCR products. dNTP can bind to Mg2+ to reduce the concentration of free Mg2+.

Template (target gene) nucleic acid: The amount and purification degree of template nucleic acid is one of the key steps in the success or failure of PCR. Traditional DNA purification methods usually use SDS and proteinase K to digest the specimen.

The main functions of SDS are: lysing lipids and proteins on the cell membrane, thus dissolving membrane proteins to destroy cell membranes and dissociating nuclear proteins in cells. SDS can also bind to proteins and precipitate; proteinase K can hydrolyze and digest proteins, especially The histone bound to the DNA is extracted with the organic solvent phenol and chloroform to extract the protein and other cellular components, and the nucleic acid is precipitated with ethanol or isopropanol. The extracted nucleic acid can be used as a template for the PCR reaction. For general clinical test specimens, the cells can be lysed by a quick and simple method, the pathogen is lysed, the protein of the chromosome is digested and the target gene is released, and it is directly used for PCR amplification. RNA template extraction is generally performed using guanidinium isothiocyanate or proteinase K to prevent RNase from degrading RNA.

Mg2+ concentration: Mg2+ has a significant effect on the specificity and yield of PCR amplification. In the general PCR reaction, when the concentration of various dNTPs is 200umol/L, the concentration of Mg2+ is preferably 1.5-2.0mmol/L. When the concentration of Mg2+ is too high, the specificity of the reaction is lowered, and non-specific amplification occurs. If the concentration is too low, the activity of Taq DNA polymerase is lowered, and the reaction product is reduced.

Selection of PCR reaction conditions: PCR reaction conditions are temperature, time and number of cycles.

Temperature and time setting: Three temperature points of denaturation-annealing-extension are set based on the three steps of the PCR principle. In the standard reaction, the three-temperature method is used, the double-stranded DNA is denatured at 90-95 ° C, and then rapidly cooled to 40-60 ° C. The primer is annealed and bound to the target sequence, and then rapidly heated to 70-75 ° C in Taq DNA. The primer strand is extended along the template by the action of the polymerase. For shorter target genes (100-300 bp in length), a two-temperature method can be used. Except for the denaturation temperature, the annealing and extension temperatures can be combined into one. Generally, the denaturation at 94 °C is used, and the annealing and extension are performed at about 65 °C. Taq DNase still has high catalytic activity).

1 Denaturation temperature and time: The denaturation temperature is low, and the incomplete melting is the most important cause of PCR failure. In general, 93 ° C ~ 94 ° C 1 min is enough to denature the template DNA, if it is lower than 93 ° C, it needs to extend the time, but the temperature can not be too high, because the high temperature environment has an effect on the activity of the enzyme. If this step does not completely denature the target gene template or PCR product, it will lead to PCR loss.

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2 annealing (refolding) temperature and time: annealing temperature is an important factor affecting PCR specificity. After the denaturation, the temperature is rapidly cooled to 40 ° C ~ 60 ° C, which can be combined with the template. Since the template DNA is much more complex than the primer, the chance of collisional binding between the primer and the template is much higher than the collision between the complementary strands of the template. The annealing temperature and time depend on the length of the primer, the base composition and its concentration, and the length of the target sequence. For 20 nucleotides, a primer with a G+C content of about 50%, 55 ° C is preferred as the starting point for selecting the optimum annealing temperature. The renaturation temperature of the primers can be used to help select the appropriate temperature by the following formula:

Tm value (melting temperature) = 4 (G + C) + 2 (A + T)

Refolding temperature = Tm value - (5 ~ 10 ° C)

Selecting a higher renaturation temperature within the allowable range of Tm values ​​can greatly reduce non-specific binding between the primer and the template, and increase the specificity of the PCR reaction. The renaturation time is generally 30 to 60 sec, which is sufficient to completely bind the primer to the template.

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