Despite the development of automated PCR cyclers, scientists still struggle to obtain optimal and efficient results. Suzanne Kennedy, co-editor of the new book PCR: Troubleshooting and Optimization, shares some insights on how to get the most out of PCR experiments.
“The top problem in the lab, most often, is contamination,” says Kennedy. “Along with that comes how to prevent it, control it, detect it, and to decipher results around it.”
Inhibitors present another challenge, according to Kennedy. These molecules prematurely end amplification and lead to confusion in qPCR data analysis. To work around inhibitors, use the inhibitor removal technology (IRT) to enhance PCR. Certain additives can help with difficult templates, such as amplification bias templates. Another trick is to dilute the sample 10%. “Most people think that if something is not amplifying, they should add more DNA; however, sometimes less is more,” she says.
Kennedy’s guide to PCR optimization also simplifies the process of qPCR data analysis. “There are just a lot of mathematical calculations involved, and people get very confused,” she says.
Over the history of PCR, researchers have developed and modified PCR techniques for specific purposes in a variety of biological research applications. For example, emulsion and droplet PCR are now widely used in second-generation sequencing applications. In the future, researchers will probably use more microfluidic PCR techniques, which amplify nucleic acids from very small sample sizes. But these techniques are not common in laboratories just yet. “People don’t have the right instrumentation to do it yet,” says Kennedy.
Whole-genome amplification could also become popular in the future. Traditional PCR amplifies small regions of the genome, which is a time-consuming and technically challenging process when using small amounts of DNA. While amplifying the entire genome alleviates these problems, it introduces others, such amplification bias. Researchers are now attempting to develop methods that combine whole genome amplification with PCR to provide efficient and accurate amplification results.
Denature, anneal, amplify, and repeat. While the basics of PCR are simple, researchers are still working on mastering the techniques and keeping up with new developments.
In addition to being co-editor for the new book PCR: troubleshooting and optimization, Kennedy is also director of research and development at Mo Bio Laboratories, Inc. (Carlsbad, CA) and a moderator of the BioTechniques Molecular Biology Forums.