The polymerase chain reaction (PCR) is one of the most common techniques employed in molecular and medicinal biology. PCR includes replicating a single-stranded DNA using DNA polymerase and synthetic primers. Since its development in 1983, PCR has become a critical component of molecular biology with applications ranging from disease diagnosis to basic research, forensic investigation, copy number analysis, pharmacodynamic testing, and agricultural testing.
PCR amplifies a single DNA molecule into several copies. This application is performed in three simple steps: denaturation, annealing, and extension. These simple steps are repeated 25 to 35 times to generate copies of the target DNA. This principle has remained the same, but different methods have been developed using innovative instruments and reaction mixtures. Some PCR methods include direct PCR, qRT-PCR analysis, digital PCR analysis, multiplex PCR, nested PCR, and many more. The current article shares a practical guide to common issues and solutions in PCR analysis.
Tips for PCR services
No or low amplification
Poor integrity of DNA templates may be due to minimizing the nicking of DNA during isolation. Researchers may evaluate template DNA dignity using electrophoresis while storing it in buffer or molecular-grade water to prevent degradation. In case of insufficient DNA template quality, study the input DNA quantity and increase the amount if necessary. Select highly sensitive DNA polymerase for amplification.
Aliquot primers after resuspension. Ideally, reconstitute fresh primers or get new primers for experiments. For insufficient quantity, optimize primer concentrations. Besides, use a minimum constant pressure of 0.5 micromolar for degenerative primer PCR and long PCR.
In case of sub-optimal denaturation, scientists must optimize the time and temperature of denaturation. Short times and low temperatures may make it challenging to separate double-stranded DNA templates. On the other hand, long times and higher temperatures may decrease enzyme activity. In sub-optimal annealing conditions, one must optimize the annealing temperature in a gradient format. Besides, use optimal buffers and annealing temperature for DNA polymerase.
Nonspecific amplification
Poor DNA templates may disappear as smears in gel electrophoresis. Hence, researchers may minimize the nicking and shearing of DNA during isolation. For complex DNA template sequences, choose DNA polymerase that displays high affinity for the DNA template. Besides, using a co-solvent or PCR additive to denature GC-rich DNA sequences can be helpful.
In case of excess DNA polymerase, review the recommended DNA polymerase amount and decrease the amount if necessary. On the other hand, for inappropriate DNA polymerase, use Hotstar DNA polymerase, which is functional after the high-temperature activation step. This step may increase the specificity of DNA polymerases.
Sequence errors within PCR products
Use exceptionally high-fidelity DNA polymerase to produce PCR fragments for applications such as sequencing, cloning, and site-directed mutagenesis. Evaluate excessive magnesium concentrations and reduce them if necessary.
A high number of PCR cycles can increase mismatched nucleotides. Hence, reduce the number of cycles without lowering the yield of PCR products. On the other hand, one may increase the input DNA amount to avoid excessive cycles.
In conclusion
PCR analysis is a robust tool with applications in medical and clinical research.
