The exults of temperature performance tests of several brands of PC cycle’s showed that most cycle’s do not perform within the manufacturers’ specifications. Furthermore, the measured temperature accuracies and spreads can have a substantial impact on the generated PC results and (diagnostic) conclusions. None of these “out of specs” cycles indicated that the temperature performance was not as “expected”, nor did any (warning) messages appear on the display of the cycle’s. Our conclusion is that laboratories performing cycling reactions (PC, Cycle-sequencing, LLC, PC) should validate their cycle 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 MATS measurement sys tem, but also supported by biological evidence . Introduction Although the PC process is a dynamic reaction, which takes place once the first denomination of the template is achieved (assuming that all needed ingredients are available in the reaction mixture), most of the PC process variables are over- simplified by the majority of users, manful utters 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 cycle impacts on amplification results like: (1) heat-block -disposable fit characteristics, (2) Applier hot and cold spots, (3) single sensor versus multi-sensor cycle technology, (4) partial block load effects, and other effects.
Simplification of the Cycling process The PC Cycling Process Denomination During denomination, the double stranded DNA melts open to single minimum. During denomination partial heat inactivation of the polymerase enzyme occurs. During denomination a subtle balance between enzyme inactivation and template denomination is established. Most of the people performing PC reactions within their laboratory have a (molecular) biology background. This background is the cause that most users (wrongly) prioritize denomination over enzyme deactivation (heat inactivation).
First, batch-to-batch variations of so called “heat stable enzymes” can be substantial. Secondly, the highest activity of these enzymes should be present during the last cycles of the process, when the largest amount of template is available. Because the enzyme deactivation (heat-inactive Zion) already took place this may result in enzyme depletion, and a significant decrease or even lack of yield. Influence of denomination temperature on enzyme depletion (and reach Zion yield) caused by enzyme heat inactivation. Standard DNA template.
Running conditions: annealing ICC, 60 s, denomination 30 s Denomination time 1 second 5 seconds Influence of denomination temperature and time on enzyme depletion, enzyme inactivation and reaction yield. In time the enzyme is heat Inca titivated due to a to high denomination temperature. Notice that the denomination temperature should not exceed 94 co, preferable ICC and the holding should normally not exceed 1 minute. Standard DNA template. Running conditions: annealing ICC, 60 s, and denomination from 1 to 60 s.
Tax Polymerase 5 units, elongation (extension) constant time. 1 5 seconds 30 seconds 60 seconds Annealing The primers are Jiggling around. Hydrogen bonds are constantly formed and broken between the single stranded primers and the single stranded template. More stable bounds last a little bit longer (primers that fit exactly) and on that little double- stranded piece the polymerase can attach and start copying the template. Annealing mimes and temperatures are important to optimize signal and to minimize non- specific binding of primers to the template.
Influence of annealing temperature on results using standard DNA template. Running conditions : denomination ICC, 15 s, annealing 15 s, Tax Polymerase 5 Units, Annealing time Influence of time on annealing temperature. Standard DNA template, running conditions: denomination ICC, 1 5 seconds, Tax Polymerase 5 units, elongation (extension) constant time.. Extension During extension the primer binds to the single stranded DNA template on the 3′ side. The polymerase adds dent’s complementary to the tem plate from 5′ to 3′, eating the template from 3′ to 5′ side.
The ideal working temperature (mostly ICC) for the polymerase is used. PC Temperature Measurement – The MATS System The above-mentioned biological tests show that that both time and correct temperature are of vital importance to generate reproducible PC results. It is therefore obvious that a cycle should perform accurate and precise, as well as in respect to targeting to the correct temperature in short time with limited overshoots and undershoots, as well as performing as uniform as possible. We have measured substantial amounts of PC cycle’s in the market.
The used measurement system (MATS) is developed by GENE-tropics, Leningrad, The Netherlands. It is the most sensitive, accurate and independent PC cycle temperature measurement system, available world wide. The system acquires temperature data with a software sampling rate of GHz, with 16 independent channels simultaneously. The data is captured dynamically and the measurements are traceable to international temperature standards (ITS-90/Nils). The MATS systems are accredited by national and international councils of temperature accreditation. The MATS system has the following specifications:
Temperature reference Number of channels Measurement resolution Measurement range Accuracy per channel Maximum deviation between channels Temperature measurement speed (software) Sensor self-heating Independent calibration steps for ITS-90 scale (traceable) 16 channels 16 bits 10 to 105 co Better than В±0. 1 co Better than 0. 05 co (uncelebrated (16 channels x 2 samples) per second Eliminated by hardware – Hardware – Probe sets – In hardware – In each multiplier set – CE-mark – Temperature accuracy validation by Dutch Council of Accreditation Accreditation The protocol The MATS protocol used for testing is described as follows: MATS DYNE-
STATS I -rest protocol: 30 co 1 minute 95 co 3 minutes 30 co 2 minutes 90 co 3 minutes 50 co 3 minutes 70 co 3 minutes 60 co 3 minutes 30 co 1 minute Sensor location pattern for the measurement of 96 x 0. 2 ml block types The test protocol covers all important temperatures of a standard PC reaction as well as the temperatures that cycle manufacturers use to specify their equipment. In some cases cycle manufacturers are incomplete in providing exact specification data. All measured cycle’s were running in block mode.