Rapid and Simple Method of qPCR Primer Design
- 6.5k Downloads
Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized “real-time” using a computer. In qPCR, a reporter dye system is used which intercalates with DNA’s region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon®, SYBR Green®, and Taqman®. However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest®, Unafold®, and Beacon designer®) to design qPCR primers.
Key wordsQuantitative real-time polymerase chain reaction qPCR Real-time PCR Primer design Free online software SYBR Green primers
- 3.D’haene B, Hellemans J (2010) The importance of quality control during qPCR data analysis. Int Drug Disc:18–24Google Scholar
- 4.Bustin S, Nolan T (2004) Pitfalls of quantitative real-time reverse transcription polymerase chain reaction. J Biomol Tech 14:155–166Google Scholar
- 5.Paudel D et al (2001) Comparison of real-time SYBR Green dengue assay with real-time TaqMan RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection. N Am J Med Sci 3(10):478–485Google Scholar