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Rapid and Simple Method of qPCR Primer Design

  • Brenda Thornton
  • Chhandak BasuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1275)

Abstract

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 words

Quantitative real-time polymerase chain reaction qPCR Real-time PCR Primer design Free online software SYBR Green primers 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Treasure Coast High SchoolPort St. LucieUSA
  2. 2.Department of BiologyCalifornia State UniversityNorthridgeUSA

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