Mastering PCR: Tips and Tricks for Successful Amplification

Mastering PCR: Tips and Tricks for Successful Amplification


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  • Source: Microbioz India

  • Date: 18 Apr,2024

A key tool in molecular biology, the polymerase chain reaction (PCR) amplifies a particular DNA fragment. It’s an effective tool for many uses, such as forensics, genetic research, and diagnosis.

Some tips and tricks for getting reliable PCR results are given below:

Create high-quality primers:

The best primers should be designed in such a way that they have foreseen melting temperature (Tm) against the target DNA sequence, lack regions with secondary structures or primer-dimer formation. Utilize online tools and software to aid in primer design and validation.

Optimize primer concentrations:

Determine which concentration of the primers is optimal for the PCR reaction by trying out various concentrations. Either much or less amount of primer can cause non-specific amplification or poor PCR yield respectively.

Use high-quality DNA templates:

Ensure that high integrity, pure DNA template, free from contaminants, degraded DNA or inhibitors that may affect PCR amplification is used in PCR.

Optimize annealing temperature:

Establish the best annealing temperature for your primers through gradient PCR or temperature optimization experiment. In particular, temperature gradients can assist in identifying at what point specific amplification becomes most efficient while at the same time minimizing non-specific amplifications.

Optimize concentration of Mg2+:

Magnesium ions (Mg2+) are essential cofactors for DNA polymerase activity. Optimize Mg2+ concentration in the PCR reaction so that enzyme activity is maximized without inhibiting amplification.

Hot-start PCRs:

Apply hot-start PCRs techniques to reduce nonspecific amplifications and enhance specificity. Hot start methods involve activation of enzymes at higher temperatures thus no activity during early setup but stringent specificity during initial cycles of PCR.

PCR enhancers addition:

To increase efficiency of GC-rich or templates prone to form secondary structures add PCR enhancers like DMSO (dimethyl sulfoxide) or betainle. In addition these additives break down secondary structures and promote primer binding.

Touchdown PCRs:

Use touchdown PCRs where annealing temperature decreases gradually over few cycles to improve specificity and minimize non-specific amplifications.

Positive and negative controls:

To check for contamination, primer-dimer formation or other issues in PCR reactions, always have positive controls (template known to amplify) and negative controls (no template or non-target template).

Systematic troubleshooting:

In case of failure in PCR, ensure that each outcome of the reaction is assessed (primers, templates, enzyme, buffer, additives) before conditions can be optimized anew.

Alternative polymerases:

High-fidelity, proofreading activity or enhanced processivity are some of the properties that alternative DNA polymerases may possess thus improving PCR performance for selected applications or difficult templates.

Thus by following these tips and tricks and carefully optimizing your PCR conditions you will increase your chances of successful amplification resulting into reliable results in your experiments.

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