What is it about?
The DNA amplification method called PCR is a ubiquitous laboratory method, and underpins many important areas such as diagnosis of inherited disease, forensic analysis (DNA fingerprinting), detection of microbes, cancer research, and so on. The key reagent for PCR is called a polymerase - these enzymes are usually derived from bacteria that live in hot pools, so they withstand constant heating and cooling needed for the PCR method. Most commercial suppliers sell enzymes that are modified to prevent them working at lower temperatures, which makes the PCR process more accurate. However, when we tested a wide range of these enzymes we found that most of them still had activity at lower temperatures, and therefore do not fit the advertised description of "hot start enzymes".
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Why is it important?
This work demonstrates that few suppliers are providing enzymes for PCR that work as they are meant to. Laboratories are paying higher prices for enzymes that are not supposed to work at lower reaction temperatures, and yet most of them do. This could lead to inaccuracies and failures in laboratory analyses, or clinical diagnostic settings. It is also rather scandalous that companies can sell these products without first testing that they do not activity prior to the heat inactivation step.
Perspectives
We discovered this phenomenon serendipitously, when we could not get a particular DNA test (called a telomere length assay) to work. We found this assay was failing because the so-called "hot start" enzymes we were using had activity at lower temperatures. We had to screen multiple commercial enzymes to find some that actually worked as they were meant to, and did not start copying DNA until the reaction reached a higher temperature. When we inspected the manufacturer's product sheets for these enzymes, we found that although the DNA polymerisation (copying) activity was measured and guaranteed, no tests or warranties were provided for the "hot start" capability. Our paper outlines a simple method to screen enzyme samples to determine whether they work as they should. This can be used by laboratories to search for the best hot start enzymes. However, we think the enzyme suppliers need to be held to account for these failings, and should actually demonstrate that their hot- start enzymes do not work until they are heat inactivated, then including this information in the specification sheet for each batch. The assay we publish here would allow them to carry out this batch testing and remove product that is not performing as it should.
Martin Kennedy
University of Otago
Read the Original
This page is a summary of: [Letter to the Editor] Many commercial hot-start polymerases demonstrate activity prior to thermal activation, BioTechniques, December 2016, Future Science,
DOI: 10.2144/000114481.
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