Maximizing PCR and RT-PCR Success - Introduction

2 PCR Brochure 10/2016 The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research (see Figure 1) are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers. Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges. Despite the fact that PCR amplification is performed routinely and that thousands of PCR protocols have been developed, researchers still encounter technical difficulties with PCR experiments and often fail to obtain specific amplification products. Although there are several different challenges (e.g., smearing, low yield, and nonspecific amplification), there are only two main reasons for PCR failure or poor results. These are the specificity of the reaction and template secondary structure. This brochure presents a discussion of the factors that influence PCR and RT-PCR specificity and template denaturation – enabling successful results. The factors discussed are: • Choice of enzyme (page 3) • Reaction conditions (i.e., choice of buffer, annealing temperature, etc) (page 7) • Automatability of PCR (page 9) • Ease of use (page 12) • Primer design (page 14) • Template quality (page 15) • Challenging applications and new technologies (page 15) Introduction Critical factors influencing PCR success Research areas Applications Biomarker discovery Pathogen identification Gene regulation research Cell development research Cancer research Drug validation/applied biomarkers Gene silencing/siRNA/miRNA) Epigenetics Gene expression analysis Genotyping Food testing Blood testing Vaccination Sample quality and amount Costs Normalization Sensitivity Reliability of results Time to result Sample collection and stabilization Nucleic acid purification Amplification Detection Challenges Methods Figure 1. Overview of research areas and associated challenges.

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