Polymerase Chain Reaction (PCR) is a fundamental molecular biology technique for gene detection and pathogen identification. However, reaction milieu inhibitors can reduce PCR assay effectiveness.
Internal components and external pollutants can hinder PCR results, affecting accuracy and reliability. This article highlights these factors in detail.
Interior PCR Factors
Initial Design
Customized DNA oligonucleotides, determine PCR primers amplification’s specificity and efficacy. PCR success depends on primer design.
The typical primer length is 15–30 nucleotides. Long primers impede template binding and may form secondary structures, while short ones reduce specificity.
Primers need 40%–60% GC. GC uniformity between forward and reverse primers is needed for balanced annealing.
Extreme GC hinders primer-template annealing. Complementary sequences must not be between or inside primers.
Random base distribution is necessary to prevent primer dimer formation, which disrupts template annealing.
Ideal Tm
Primer-template pairs melt around 55–80°C. Balanced annealing within 5°C is achieved by Tm uniformity between forward and reverse primers.
Primers should be designed from genomic regions with few secondary structures to speed up amplification.
Quality
Oligos should couple 99% or more. Quality can be improved using RPC, PAGE, or HPLC impurity removal. High coupling efficiency allows the synthesis of 200-base oligos with research-specific modifications.
PAGE and Maldi-Tof Mass QC validate oligo purity and reliability for PCR. These steps improve molecular biology accuracy and reproducibility.
Enzyme concentration
High concentrations produce nonspecific amplification, while low amounts reduce product synthesis.
Nucleic acid template
Quantity, purity, and integrity of nucleic acid affect PCR.
PCR
There are three temperature and time points:
These are denaturation, annealing, and extension. Templates with high GC require longer denaturation.
For PCR specificity, raise the annealing temperature if the primer has few bases and lower it if it has many bases to assist it bind to the DNA template. Longer products require longer extensions.
Cycles
Template DNA concentration determines PCR amplification cycle count. Cycles increase nonspecific products and base misincorporation.
External Factors in PCR
- SDS: An ionic detergent that disrupts enzyme proteins’ non-covalent connections (hydrogen bonds and hydrophobic interactions), denaturing and destroying their shape and function. 0.01% completely inhibits PCR, while 0.005% considerably lowers yield.
- The organic solvent phenol denatures enzymes. PCR yields are considerably lowered at 0.2% and completely inhibited at 0.5%.
- Organic solvent ethanol inhibits PCR above 1%.
- Isopropanol inhibits PCR better than ethanol.
- PCR reactions are inhibited by sodium acetate (NaAc) at concentrations higher than 5 mM. causes precipitation by lowering the pH of the reaction system and creating sodium salt complexes with DNA.
- Higher animals’ blood, liver, and muscle color proteins contain hemoglobin’s prosthetic group, heme pigment. PCR stops above 1mg/ml. Organic solvent DNA extraction is challenging, leaving residue.
- Hemin Chloride: Hematin’s primary ion, divalent iron ion, is hemin chloride. When heated and exposed to oxidants, it can oxidize to a trivalent iron ion, which is known as brown hemin chloride. When the concentration is higher than 0.1ng/μl, it inhibits PCR processes.
- Urea—carbon, nitrogen, oxygen, and hydrogen—comes from urine. PCR inhibitor over 20mM. Protein metabolism creates urea, which kidneys filter into urine.
- Plants naturally contain tannic acid, commonly referred to as tannin acid, which is a phenolic organic chemical. When the concentration is higher than 0.1ng/μl, it inhibits PCR processes.
- Natural water, soil, and plants produce humic acid. Humic acid, a black-brown organic substance produced by microbial degradation of soil dead materials, comprises plant development and fertility components. It comprises 50-70% soil organic materials. Humic acid contains organic compounds, principally humic and fulvic acids, with comparable and different compositions, structures, and properties.
- Hemoglobin and its metabolites may severely impair Taq enzyme activity and PCR amplification. Iron ion release may impede.
- Hyperlipidemia makes Real-Time PCR difficult by shielding or absorbing fluorescence from blood low-density lipoproteins. Hyperlipidemia reduces fluorescence and signal. Centrifugation at low temperature and high speed eliminates lipemic interference.
Conclusion
Understanding and regulating internal and external PCR variables is essential for molecular biology experiment reliability.
Scientists can improve PCR-based tests by following primer design principles, assuring primer and enzyme quality, optimizing reaction conditions, and minimizing inhibitor effects.
To prevent internal folding, the primer should have a comparatively basic structure and no internal secondary structures.
Primer-primer annealing, which produces primer dimers and interferes with the amplification process, must also be avoided.
When designing, if one is uncertain which nucleotide to place at a specific location in the primer, they might include multiple nucleotides at that location, which is known as a mixed site.
Additionally, for more extensive pairing capabilities, a nucleotide-based molecular insert (inosine) can be used in place of a conventional nucleotide.
