Primer Optimization for SNP Based PCR And Quantitative Real Time PCR Assays

Abstract

Primer designing is critical for efficient polymerase chain reaction (PCR) and real time PCR, especially for quantification of real time PCR assays and single nucleotide polymorphism (SNP) for genotype analysis. Careful selection and optimization of primers is necessary for specific, reliable and efficient results in these methodologies. While detecting SNP, it is important for primers to amplify specifically the target region with minimal risk of non-specific amplification or primer dimer formation. In real-time PCR or quantitative PCR (qPCR), primer designing is more complex as primers must be compatible with both the target sequence as well as fluorescent reporter system used to monitor the amplification in real-time. Therefore, knowledge and understanding of primer designing and principles of optimization are necessary for researchers and analysts who are dependent on PCR-based methods for required results. This review provides a comprehensive overview of the key considerations to design primers in SNP-based PCR and real-time PCR assays, covering the important aspects such as primer length, melting temperature, GC content, and specificity. It also elaborates the use of specialized software tools for primer designing and the importance of experimental validation to ensure optimal assay performance.