Factors Affecting PCR Test Results
1. Primers:
Primers are the key to specific reactions in PCR, and the specificity of PCR products depends on the degree of complementarity between primers and template DNA.
Primers should be designed according to the following principles:
1) Primer length: 15-30 bp, usually around 20 bp.
(2) Primer amplification span: 200-500 bp is appropriate, and fragments up to 10 kb can be amplified under specific conditions.
(3) Primer bases: G+C content should be 40-60%. If G+C is too little, the amplification effect will not be good, and if G+C is too much, non-specific bands will easily appear. ATGC is preferably randomly distributed, avoiding clusters of more than 5 purine or pyrimidine nucleotides.
(4) Avoid secondary structures inside the primers, and avoid complementarity between two primers, especially the complementarity at the 3' end, otherwise primer dimers will be formed and non-specific amplification bands will be generated.
(5) The bases at the 3' end of the primer, especially the last and penultimate bases, should be paired strictly to avoid PCR failure due to terminal base mismatch.
(6) There are or can be added suitable enzyme cutting sites in the primers. The target sequence to be amplified should preferably have appropriate enzyme cutting sites, which is very beneficial for enzyme analysis or molecular cloning.
(7) Specificity of primers: Primers should have no obvious homology with other sequences in the nucleic acid sequence database. Primer amount: The concentration of each primer is 0.1-1 umol or 10-100 pmol. It is better to use the lowest primer amount to produce the required results. A high primer concentration will cause mismatch and non-specific amplification, and it can increase the amount of primers. Opportunity to form dimers.
2. Enzyme and its concentration
There are currently two kinds of Taq DNA polymerase available, one is a natural enzyme purified from Thermus aquaticus, and the other is a genetically engineered enzyme synthesized by Escherichia coli. About 2.5 U of enzyme is needed to catalyze a typical PCR reaction (when the total reaction volume is 100 ul). If the concentration is too high, it will cause non-specific amplification, and if the concentration is too low, the amount of synthetic product will decrease.
3. Quality and concentration of dNTP
The quality of dNTP is closely related to the concentration and PCR amplification efficiency. The dNTP powder is in the form of granules, and if it is improperly stored, it will become volatile and lose its biological activity. The dNTP solution is acidic, and should be prepared at a high concentration before use. Adjust its pH to 7.0-7.5 with 1 M NaOH or 1 M Tris.HCL buffer solution, aliquot it in small quantities, and store it in a freezer at -20°C. Multiple freeze-thaw cycles will degrade dNTPs. In the PCR reaction, the dNTP should be 50-200 umol/L, especially note that the concentrations of the four dNTPs should be equal (equal molar preparation), if the concentration of any one of them is different from the others (high or low) , causing a mismatch. If the concentration is too low, the yield of PCR products will be reduced. dNTP can combine with Mg2+ to reduce the concentration of free Mg2+.
4. Template (target gene) nucleic acid
The amount and purification degree of template nucleic acid is one of the key links in the success or failure of PCR. Traditional DNA purification methods usually use SDS and proteinase K to digest and process specimens. The main function of SDS is to dissolve the lipids and proteins on the cell membrane, thereby dissolving the membrane protein and destroying the cell membrane, and dissociating the nuclear protein in the cell. SDS can also bind to protein and precipitate; proteinase K can hydrolyze and digest protein, especially Histones bound to DNA are extracted with organic solvents phenol and chloroform to remove proteins and other cellular components, and nucleic acids are precipitated with ethanol or isopropanol. The extracted nucleic acid can be used as a template for PCR reaction. For general clinical testing specimens, a fast and simple method can be used to lyse cells, lyse pathogens, digest and remove chromosome proteins to free target genes, and directly use them for PCR amplification. RNA template extraction generally uses guanidine isothiocyanate or proteinase K method to prevent RNase from degrading RNA.
5. Mg2+ concentration
Mg2+ has a significant impact on the specificity and yield of PCR amplification. In general PCR reactions, when the concentration of various dNTPs is 200 umol/L, the appropriate Mg2+ concentration is 1.5-2.0 mmol/L. If the concentration of Mg2+ is too high, the specificity of the reaction will be reduced, and non-specific amplification will occur. If the concentration is too low, the activity of Taq DNA polymerase will be reduced, and the reaction product will be reduced.
6. Setting of temperature and time
Based on the three steps of PCR principle, three temperature points of denaturation-annealing-extension are set. In the standard reaction, the three-temperature point method is used. The double-stranded DNA is denatured at 90-95°C, and then rapidly cooled to 40-60°C. The primer anneals and binds to the target sequence, and then rapidly heated to 70-75°C. Under the action of polymerase, the primer strand is extended along the template. For shorter target genes (when the length is 100-300 bp), the two-temperature point method can be used. In addition to the denaturation temperature, the annealing and extension temperatures can be combined into one. Generally, 94°C is used for denaturation, and about 65°C for annealing and extension (this Temperature Taq DNase still has high catalytic activity).
1) Denaturation temperature and time: Low denaturation temperature and incomplete melting are the main reasons for PCR failure. Generally, 93°C~94°C for 1 min is enough to denature the template DNA. If it is lower than 93°C, the time needs to be extended, but the temperature should not be too high, because the high temperature environment will affect the activity of the enzyme. If this step cannot completely denature the target gene template or PCR product, it will lead to PCR failure.
(2) Annealing (refolding) temperature and time: Annealing temperature is an important factor affecting PCR specificity. After denaturation, the temperature is rapidly cooled to 40°C-60°C to allow primers and templates to combine. Since template DNA is much more complex than primers, the chances of collisional binding between primers and templates are much higher than those between complementary strands of templates. The annealing temperature and time depend on the length of the primer, its base composition and its concentration, and the length of the target base sequence. For primers with 20 nucleotides and a G+C content of about 50%, 55°C is an ideal starting point for selecting the optimum annealing temperature. The annealing temperature of the primer can be used to help choose the appropriate temperature by the following formula:
Tm value (melting temperature) = 4 (G + C) + 2 (A + T)
Refolding temperature = Tm value - (5~10°C)
Within the allowable range of the Tm value, choosing a higher annealing temperature can greatly reduce the non-specific binding between the primer and the template and improve the specificity of the PCR reaction. The annealing time is generally 30-60 sec, which is enough for the complete combination between the primer and the template.
(3) Extension temperature and time: Biological activity of Taq DNA polymerase:
70~80℃ 150 nucleotides/S/enzyme molecule
70℃ 60 nucleotides/S/enzyme molecule
55℃ 24 nucleotides/S/enzyme molecule
When the temperature is higher than 90°C, DNA synthesis can hardly proceed.
The extension temperature of the PCR reaction is generally selected between 70~75°C, and the commonly used temperature is 72°C. Too high extension temperature is not conducive to the combination of primers and templates. The time of PCR extension reaction can be determined according to the length of the fragment to be amplified. Generally, for DNA fragments within 1 Kb, an extension time of 1 min is sufficient. It takes 3-4 minutes for a 3-4 kb target sequence; it takes 15 minutes to amplify a 10 Kb target sequence. Excessively long extensions will lead to the appearance of non-specific amplification bands. The extension time is slightly longer for the amplification of low concentration template.
7. Number of cycles
The number of cycles determines the extent of PCR amplification. The number of PCR cycles depends mainly on the concentration of template DNA. Generally, the number of cycles is selected between 30 and 40 times, and the more the number of cycles is, the more the amount of non-specific products will increase.
8. Factors Affecting PCR Specificity
Through the above content, it can be seen that there are many factors that can affect the specificity of pcr, here we make a summary for your reference:
(1) Stringency of the annealing step: Increasing the annealing temperature can reduce mismatched hybridization and thus improve specificity.
(2) Shortening the annealing time and extension time can reduce false initiation and false extension.
(3) Primer dimer is the most common by-product, reducing the concentration of primers and enzymes can also reduce false priming, especially primer dimerization.
(4) Changing the concentration of MgCl2 can improve the specificity, which may be a direct effect on the stringency of the reaction or on the taq enzyme.
It is generally believed that PCR products should be detected by electrophoresis within 48 hours, and some of them are best detected by electrophoresis on the same day. After 48 hours, the band pattern will appear irregular or even disappear.
