Considerations for a Successful PCR Set Up

Learn about other PCR components-beyond the polymerase-that are essential for optimal results.
While the type of DNA polymerase and buffer you choose play a critical role in the success of a PCR, there are other crucial factors to consider while optimizing the PCR assay to achieve better results. These components include the template that contains the target sequence, the forward and reverse primers, dNTPs and optional additives to help boost your PCR.
The quality of the template in terms of its complexity can play a crucial role in the outcome of the PCR. For example, a GC-rich template has significantly more hydrogen bonds than an AT-rich template. This can result in incomplete strand separation or secondary structure formation that inhibits the polymerase and give rise to pre-mature termination. To overcome this, scientists can add secondary structure destabilizers such as DMSO or betaine to their reactions.
The template amount can influence the yield and efficiency of the reaction. Based on the complexity, various template amounts are recommended. Very low or high DNA concentrations may require specific modifications such as changing the number of cycles or redesigning the primers.
Having a good primer pair can dictate the PCR outcome. General recommendations to consider while designing the primers are: primer length, balanced distribution of GC- and AT-rich domains, and the annealing temperature. Additionally, it is crucial to avoid complementarity between the forward and reverse primers to prevent primer-dimer formation.
The recommended primer concentration is between 0.1 to 0.6 uM, as higher concentrations may promote mispriming and accumulation of non-specific products and very low concentrations may result in lower product yields.
In most common PCR applications, the final nucleotide concentration is recommended to be between 0.05-0.5mM. A higher concentration is preferred for long range PCR to improve the yield, while a lower concentration is recommended for increased fidelity when using non-proofreading polymerases.
Another factor to consider is the magnesium ion concentration in the reaction mixture as they bind dNTPs and reduce their availability.
Scientists may add other optional additives to enhance the PCR's efficiency and specificity. Certain additives, such as betaine and DMSO, help overcome complex secondary structures, thereby increasing the PCR amplification yield. Other additives reduce non-specific priming and off-target DNA amplification. For example, formamide is most commonly used to improve PCR specificity. Depending on your assay conditions, choose the right kind of PCR additive to boost your PCR.
Learn more at sigmaaldrich.com/pcr.
Follow The Scientist!
www.the-scientist.com
Facebook: / thescientistmagazine
Twitter: / thescientistllc
LinkedIn: / the-scientist
Instagram: / the_scientist_magazine