Influence of PCR consumables on the accuracy of real-time PCR experiments and NGS sample preparation

Gerrit Gutzke, Hanna Oldfield *, Emily Flowers
14titude Ltd, Germany 


Classical PCR and qPCR plates are one-component plates made out of polypropylene (PP). PP is the best plastic material for PCR tubes as it is chemically inert and allows for the production of ultra- thin tube walls which is important for fast temperature transfer. While PP has become the standard material for PCR consumables some of its properties question its suitability for applications like qPCR and NGS:
The material characteristics of PP exhibit a Vicat Softening Tem- perature (VST) of 90◦C and a coefficient of thermal expansion of 180 × 10−6 K−1 which are potential weaknesses for its usage at typical (q)PCR temperatures.
When used for (q)PCR, not only do the plates soften during the denaturation step, but measurements also show that the plates expand by up to 2 mm in the diagonal plane (from room temperature to 95 ◦ C) and they shrink again as the temperature decreases. Therefore, the plate will undergo expansion and contraction in every cycle, placing significant tension on the plate seals. As a result, contact between the seal and plate will be particularly weakened in the corner positions and outer rows leading to evaporation from the plate in these areas, while centre wells will only be affected minimally. This differential evaporation effect is especially eminent when adhesive seals are used (as opposed to heat seals).
Evaporation has a significant effect on the reaction conditions resulting in noticeable effects, especially for qPCR. Identical sam- ples can exhibit significant differences in their Ct values, depending on their position on the plate. This often remains unnoticed as trip- licates are typically placed in neighbouring wells which are affected by similar levels of evaporation.
A solution to the problem of evaporation related qPCR inaccu- racies is the usage of two-component plates. These plates consist of tubes made out of PP but a frame made out of polycarbonate (PC). PC does not show significant temperature-dependant expan- sion and contraction as the VST of this material is 147◦C and the coefficient of thermal expansion is 70 × 10−6 K−1 .
The ongoing trend to continually reduce DNA concentrations can make additional modifications or choices necessary. DNA has been shown to bind to PP tubes in trace amounts, especially in highly ionic conditions. Different PP polymers are used for the pro- duction of PCR consumables as they differ in their characteristics, such as surface charge. As a result, different PCR plates bind DNA in varying amounts. Furthermore, the commercially used term “low- binding” is poorly defined.
DNA binding to PP surfaces has not been reported as an issue for PCR/qPCR as DNA stuck to the walls will be released during denaturation steps. However, NGS protocols contain a number of transfer steps from one tube to another and ultra-low DNA binding characteristics may be required if only trace amounts of nucleic acids are used.

Back to MIQE QC
Bookmark the permalink.

Comments are closed.