The invention of the polymerase chain reaction (PCR) by K. Mullis and co-workers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation, and cancer research, are challenging today's PCR technologies with more demanding requirements. These include the need for increased throughput, higher assay sensitivity, and reliable data analysis. Assay development and evaluation, reproducibility of data, and time to result are still major problems encountered by researchers.
PCR amplification is performed routinely and thousands of PCR protocols have been developed, yet 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 two main reasons for PCR failure or poor results: the specificity of the reaction and template secondary structure.
PCR is both a thermodynamic and an enzymatic process. Successful real-time PCR requires amplification and detection under optimal conditions and each reaction component can affect the result. The annealing step is critical for high PCR specificity. When primers anneal to the template with high specificity, this leads to high yields of specific PCR products and increases the sensitivity of the amplification reaction. However, due to the high primer concentration in the reaction, primers will also hybridize to non-complementary sequences with mismatches. If the primers anneal to the template sequence with low specificity, amplification of nonspecific PCR products and primer–dimers may occur. Competition in the amplification reaction between these artifacts and the desired PCR product may reduce the yield of the specific product, thereby reducing the sensitivity and linear range of the real-time reaction. Low PCR specificity can significantly affect quantitative PCR particularly when using SYBR Green for detection. As SYBR Green binds to any double-stranded DNA sequence, primer–dimers and other nonspecific PCR products will generate a fluorescent signal. This reduces the overall sensitivity of the assay and also leads to inaccurate quantification of the transcript of interest. Factors critical for high specificity in PCR include primer design and the reaction chemistry used.