Improving Real-Time PCR Success

Introduction

To meet increasing food supply demands of the global population, growers require seeds that can withstand the regional challenges found in their fields. In order to develop crop varieties that are more resistant to insects, disease and environmental stressors, breeders use genotypic information to improve breeding program decisions.

Situation

A multinational seed company using high-throughput real-time PCR to genotype crop samples was experiencing higher-than-expected amplification failures. Unable to allocate full-time resources to troubleshoot their protocol, the company chose Sigma-Aldrich to help resolve their amplification issues and increase their real-time PCR success rate.

Solution

Sigma-Aldrich partnered with the company’s genotyping lab to evaluate their reagents and amplification parameters. The company's genotyping protocol was replicated in a Sigma-Aldrich lab and the reported amplification failures were observed. Next, reagents were individually evaluated and it was determined that amplification failures correlated with the company's extraction solution.

Each component of the extraction solution was then separately titrated into individual PCR reactions and one component was found to strongly inhibit sample amplification. The company previously suspected the presence of unidentified PCR inhibitors as a possible cause of failed amplification and had added a step to their protocol to dilute extracted samples. Sigma-Aldrich discovered that the dilution volume the company was using to eliminate suspected inhibition left many samples with insufficient DNA to produce an amplification signal below the background threshold of their assays.

With this insight, Sigma-Aldrich's R&D agriculture team re-engineered the company's genotyping protocol by reformulating their extraction solution, reducing their dilution factor and making complimentary modifications to their PCR master mix. These changes showed a 10-fold improvement in real-time PCR efficiency and achieved the company's target real-time PCR success rate.

A Focus On Agriculture

At Sigma-Aldrich, our experience and dedicated agricultural resources, combined with our global reach, make us the ideal partner to help you achieve your goals. We have the tools, products and systems in place that allow you to create the solutions that growers everywhere need to help feed the world.

Explore How We Can Improve Your Workflow

Visit sigma-aldrich.com/agriculture

Materials