Guidelines for Polymerase Chain Reaction (PCR)

PCR

Polymerase Chain Reaction (PCR) is a method that revolutionized molecular biology and molecular medicine. As a crucial tool in diverse research areas such as biomarker discovery, gene regulation and cancer research, PCR has widespread applications. Nevertheless, the increasing demands on PCR technology present challenges, including the need for higher throughput, improved assay sensitivity and more reliable data analysis. Researchers continually confront issues related to assay development, reproducibility of results, and the speed at which results are obtained.

PCR Overview

PCR technologies are continuously challenged by the increasing complexity of scientific research. The technique is essential for advancing major research areas by meeting the demands for higher throughput and more sensitive assays. Despite its widespread use and the development of numerous protocols, PCR experiments often do not yield specific amplification products due to technical difficulties. The evolving requirements of fields like cancer research and gene regulation push today's PCR technologies to their limits, necessitating continual improvements in assay sensitivity and data reliability.

PCR Amplification

PCR amplification is a routine procedure in laboratories worldwide, with thousands of protocols developed to date. Yet, achieving specific and reliable PCR amplification remains challenging due to factors such as reaction specificity and the secondary structure of the template. The high specificity of primers during the annealing step is crucial for obtaining high yields of specific PCR products, enhancing the amplification reaction's sensitivity. However, the high concentration of primers often leads to their hybridization with non-complementary sequences, resulting in nonspecific amplification products and primer dimers. These artifacts can compete with the desired PCR product, reducing both yield and sensitivity, particularly in quantitative PCR applications.

PCR Process

The PCR process is both thermodynamic and enzymatic, requiring optimal conditions for successful real-time PCR amplification and detection. Each reaction component, from primer design to reaction chemistry, is critical in achieving high PCR specificity. Low specificity can significantly impact the assay, especially when using detection methods like SYBR Green, which binds to any double-stranded DNA and can generate signals from nonspecific products. This reduces the assay's sensitivity and leads to inaccurate quantification of the target transcript. Therefore, optimizing PCR conditions and primer design is essential for minimizing the impact of nonspecific artifacts and improving the overall quality and accuracy of PCR results.