A PCR reaction process is divided into many steps and is also influenced by many factors, which can be divided into internal and external factors, internal factors being the components of the normal reagent system and external factors being environmental or sample carry-over factors.
What are the internal factors that affect the PCR reaction?
Primers:
Primers are the most important part of the whole PCR system and the key to determine the specificity of a PCR reaction. Primers should follow certain principles (length, amplification span, bases, etc.) when designing.
Enzyme and its concentration:
There are two Taq DNA polymerases, one is a natural enzyme purified from Bacillus subtilis and the other is a genetically engineered enzyme synthesized by Escherichia coli, too high concentration of enzyme can cause non-specific amplification, too low concentration will reduce the amount of synthetic products.
The quality and concentration of dNTP:
dNTP solution is acidic, generally configured into the system needs to be adjusted to 7.0-7.5, under normal circumstances the concentration of the four dNTP should be equal, if one of them is too high will cause a mismatch, and the concentration is too low will cause the yield of PCR products.
Template nucleic acid (target):
The amount of template nucleic acid and the degree of purification is one of the key aspects of the success or failure of PCR, and for RNA template more attention should be paid to the degradation of RNA.
Mg2+ concentration:
It mainly affects the specificity and yield of PCR amplification and generally corresponds to the concentration of dNTP. too high Mg2+ concentration will reduce the specificity of the reaction and non-specific amplification will occur, too low concentration will reduce the activity of Taq DNA polymerase and make the reaction products decrease.
Temperature and time settings:
The three steps of the PCR principle involve three temperature points: denaturation-annealing-extension, that is, denaturation temperature and time, annealing (denaturation) temperature and time, and extension temperature and time.
Number of cycles:
The number of cycles determines the degree of PCR amplification, and the number of PCR cycles depends mainly on the concentration of template DNA. The general temperature cycle number is between 30-40 times, the more cycles, the more the amount of non-specific products.
What are the external factors that affect the PCR reaction?
SDS: ionic decontamination agent, 0.01% can completely inhibit PCR reaction, 0.005% can significantly reduce the yield
Phenol: 0.5% can completely inhibit the PCR reaction, 0.2% can significantly reduce the yield
Ethanol: greater than 1% concentration can inhibit PCR reaction, but in some systems can increase the yield
Isopropyl alcohol: slightly stronger inhibition than ethanol
Sodium acetate: greater than 5 mM inhibited PCR reaction
Sodium chloride: greater than 25mM inhibits PCR reaction (saline has no effect)
EDTA:0.5mM can make PCR products reduce, 1mM no PCR products at all
Hemoglobin (heme) - source: blood, greater than 1mg/ml inhibition of PCR reactions
Heme: greater than 0.1ng/μl inhibits PCR reaction
Tannic acids: >0.1ng/μl to inhibit PCR reaction
Heparin: >0.15IU/ml inhibits PCR reaction
Urea-source: urine, inhibition at >20mM
Humic substances-source: soil, plant material, natural water
Plant polysaccharides-source: feces, plant polysaccharides
Polystyrene, polypropylene - source: UV irradiated plastic tubes
Bile acid salts - source: feces
Collagen - source: tissues
Indigo dye - source: coarse twill cotton, jeans
Protease - source: milk
Calcium ions - source: milk, bones
Iron ions - source: blood
Xylenolamine
Bromophenoland
Bilirubin (bilirubin)
Other inhibitors
In addition to the above inhibitors, certain PCR enhancers exist, such as betaine, dimethyl sulfoxide, formamide, glycerol, PEG, spermidine and single-stranded DNA-binding protein, but at high concentrations, they also inhibit PCR, so how the enhancers are used to eliminate the effect of inhibitors and how much concentration is used have a great impact on the results.
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