Reinventing PCR: What?

What are the challenges for developing multiplex PCR assays?

The three major difficulties are: incompatibility, high background, and poor reproducibility.

The first problem (incompatibility) makes assay development very difficult; the second problem (high background) makes automation very difficult; and the third problem (poor reproducibility) makes regulatory approval very difficult. These are the reasons why we still do not see many multiplex molecular assays on the market.

First, the primer incompatibility issue. Each targets need one pair of primers to amplify. Yet each primer has its own “optimal” condition (temperature, pH, iron concentration, etc). To make two primers work together is already a challenge. The more primers get involved, the harder to find a “common ground” . Incompatibility make some targets amplified better than the other in a reaction, causing interference and introduce amplification bias.

The purpose of amplification is usually for the downstream detection steps. Detection need label (so that our eyes or instrument can see the DNA). But with conventional multiplex PCR, high concentration of labeled primers will form dimmers, yield non-specific products, lead to high noise levels for the detection steps. These noises need to be removed by many washing and purification steps. These steps are not big problems for academic labs, but for commercial  assay development, these are major problems. These noise-reduction steps are very difficult to automate, and utomation is the key for a successful commercial product.

Because of the first two problems mentioned above, it is very difficult for a manufacture to produce multiplex PCR assays. The quality control issue is too difficult to address, and the performance of the tests could not be guaranteed.

Professors reviewing papers or writing grants may not know these difficulties. Many of them just assumed that multiplex is easy.

So, there is a Need to reinvent PCR, and there is challenges, the next step is to get it done.

* Disclaimer*

Although we use the words “Diagnosis” or “Diagnostics” in this blog, our products are NOT FDA approved for such uses. We are developing technologies that may eventually have clinical applications, but for now,  all our products are for research use only.