Estimation of copy number using SYBR Green: confounding by AT-rich DNA and by variation in amplicon length

Am J Trop Med Hyg. 2008 Dec;79(6):887-92.

Abstract

Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P<0.001) and amplicons with greater AT content (P<0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / chemistry
  • Animals
  • Anopheles
  • Base Sequence
  • Benzothiazoles
  • DNA / chemistry
  • DNA, Protozoan / chemistry*
  • Diamines
  • Female
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Humans
  • Organic Chemicals / chemistry*
  • Plasmodium falciparum / genetics*
  • Polymerase Chain Reaction / methods*
  • Quinolines
  • Regression Analysis
  • Sensitivity and Specificity
  • Thymine / chemistry

Substances

  • Benzothiazoles
  • DNA, Protozoan
  • Diamines
  • Fluorescent Dyes
  • Organic Chemicals
  • Quinolines
  • SYBR Green I
  • DNA
  • Adenine
  • Thymine