The Impact of the Temperature Performance of Thermal (Pcr) Cyclers on the Generated Results, and the Obligation for Regular Validation of Pcr Thermal Cyclers
The Impact of the Temperature Performance of Thermal (PCR) Cyclers on the Generated Results, and the Obligation for Regular Validation of PCR Thermal Cyclers
Tom Hendrikx**, Marc Verblakt*, Roger Pierik** * GENO-tronics BV, Landgraaf, The Netherlands ** CYCLERtest BV, Landgraaf, The Netherlands
Abstract
A fast, powerful, and extremely sensitive multi-channel temperature measurement system was developed for the dynamic temperature validation of thermal cylers. The results of temperature performance tests of several brands of PCR cyclers showed that most cyclers do not perform within the manufacturers' specifications. Furthermore, the measured temperature accuracies and spreads can have a substantial impact on the generated PCR results and (diagnostic) conclusions. None of these "out of specs" cylers indicated that the temperature performance was not as "expected", nor did any (warning) messages appear on the display of the cyclers. Our conclusion is that laboratories performing cycling reactions (PCR, Cycle-sequencing, LCR, PCR) should validate their cycler at least twice a year, or every 100 runs. However, a more frequent validation is prefer able. The impact of sample block temperature in-uniformities and the lack of accuracy is not only proven by the used MTAS measurement sys tem, but also supported by biological evidence .
Introduction
Although the PCR process is a dynamic reaction, which takes place once the first denaturation of the template is achieved (assuming that all needed ingredients are available in the reaction mixture), most of the PCR process variables are over-simplified by the majority of users, manufac turers and others, to basically three major steps. Unfortunately, at least half of the cycling-process takes place in the temperature changes between these three steps. The authors will, despite the availability of hard evidence within their companies, refrain from showing other cycler impacts on amplification results like: (1) heat-block
Tom Hendrikx**, Marc Verblakt*, Roger Pierik** * GENO-tronics BV, Landgraaf, The Netherlands ** CYCLERtest BV, Landgraaf, The Netherlands
Abstract
A fast, powerful, and extremely sensitive multi-channel temperature measurement system was developed for the dynamic temperature validation of thermal cylers. The results of temperature performance tests of several brands of PCR cyclers showed that most cyclers do not perform within the manufacturers' specifications. Furthermore, the measured temperature accuracies and spreads can have a substantial impact on the generated PCR results and (diagnostic) conclusions. None of these "out of specs" cylers indicated that the temperature performance was not as "expected", nor did any (warning) messages appear on the display of the cyclers. Our conclusion is that laboratories performing cycling reactions (PCR, Cycle-sequencing, LCR, PCR) should validate their cycler at least twice a year, or every 100 runs. However, a more frequent validation is prefer able. The impact of sample block temperature in-uniformities and the lack of accuracy is not only proven by the used MTAS measurement sys tem, but also supported by biological evidence .
Introduction
Although the PCR process is a dynamic reaction, which takes place once the first denaturation of the template is achieved (assuming that all needed ingredients are available in the reaction mixture), most of the PCR process variables are over-simplified by the majority of users, manufac turers and others, to basically three major steps. Unfortunately, at least half of the cycling-process takes place in the temperature changes between these three steps. The authors will, despite the availability of hard evidence within their companies, refrain from showing other cycler impacts on amplification results like: (1) heat-block