Polymerase chain reaction (PCR) uses enzymes to synthesize copies of specific DNA sequences of interest in many different industries such as biotechnology, pharmaceutical, clinical diagnostics, academic, research, and agriculture, to name a few. Many factors affect the quality of PCR results, including the sample, instruments, workflow, reagents, and PCR plastics. Optimizing protocols and instruments, including PCR plastics, facilitates accurate PCR results and subsequent analysis.
PCR plastics can affect the outcome of biological experiments through its physical dimensions, method of manufacturing, and materials of construction. PCR plastics should support amplification of nucleic acids and detection without interacting with samples or reagents. Wells must be chemically inert and appropriately designed for the heating and cooling process of a PCR reaction and readout during analysis.
As assay volumes become smaller, the manufacturing preciseness and materials of construction for PCR plastics become more critical. Precision injection molding is required to produce PCR plates and tubes to highly precise dimensions. Injection molding assures that volumes and inner geometries remain consistent from plate to plate and between individual wells. PCR plastics, such as plates or tubes, are used in a PCR thermal cycler or real-time PCR instrument and must meet specific dimensions to fit the instrument block perfectly.
Many reagent companies outsource manufacturing of PCR plastics to precision injection molding facilities. An issue with outsourcing manufacturing arises if there are design or manufacturing issues that affect quality of PCR plastics as significant time may pass before the problem is solved or even recognized. By then, hundreds or thousands of defective plates may already be in customers’ hands. Recognizing the importance of addressing production issues near-real-time, Bio-Rad maintains in-house PCR plate production and operates it under international quality standards (ISO 13485). This allows for a faster response should manufacturing issues arise. In-house production also affords Bio-Rad the ability to meeting changing customer needs by producing custom PCR plates.
Materials and methods of construction profoundly affect PCR results, yet not all manufacturers fully appreciate this. Many plates available today are molded in one piece, using one type of material, often polypropylene. Bio-Rad’s Hard-Shell PCR plates feature a two-component design. The plate skirt and deck, are produced from a rigid, thermostable polymer to prevent problems like warping that may arise from high temperature changes during PCR, which can impact the effectiveness of robotic plate handling. This rigid Hard-Shell prevents distortion and shrinkage that often occurs when single-component polypropylene plates are exposed to the high temperatures of thermal cycling or heat sealing.
By contrast, thin-walled wells of a PCR plate or tubes must be inert, thermo-conductive, and uniform in thickness. Virgin polypropylene is the material of choice for this application, for its chemical and biological inertness. PCR plates should be manufactured in a clean room environment and be certified free of DNase, RNase, and human DNA.
PCR plates and tubes are specifically designed with V-shaped polypropylene wells, which are for optimal thermal transfer and sample recovery. This shape allows for the thin-walled wells to sit pressed up against the PCR instrument block which allows for precise temperature control of the sample. Sample recovery becomes very important when needing the PCR reaction for a downstream application and the v-bottom wells aid in recovery of the entire sample. Raised well rims on a PCR plate also have a vital role in preventing evaporation of the sample, leading to better sample recovery. The raised rims on a PCR plate surrounding each well must also be inert and compatible with sealing methods based on pressure, adhesives, or heat.
PCR plates and tubes are available in a range of colors to assist in process or plate identification, but there is more to color than mere appearance. While the color of a well does not affect PCR performance, it may produce anomalies in real-time PCR (qPCR). A real-time PCR instrument detects the fluorescence level of a qPCR sample as it amplifies during each cycle. When using a qPCR instrument, light scattering is common with clear plates and tubes, both from other wells and refraction within the well from the block. Using white opaque plastics for qPCR reduces light scattering and prevents fluorescence refraction from the block in the wells allowing for accurate signal detection, thus improving signal to noise. When unsure of plastic well color requirements, users can look for guidance from the qPCR instrument manufacturer.
To help aid users in selecting the correct PCR plastics for their instruments and applications, Bio-Rad has made an interactive PCR Plastics Selector. Through this tool, users can select designs and colors to meet their PCR experiment needs and identify the best PCR plate for their instrument.