Molecular Biotechnology

, Volume 53, Issue 1, pp 19–28 | Cite as

A Dual-Mode Single-Molecule Fluorescence Assay for the Detection of Expanded CGG Repeats in Fragile X Syndrome

  • Brian Cannon
  • Cynthia Pan
  • Liangjing Chen
  • Andrew G. Hadd
  • Rick RussellEmail author


Fragile X syndrome is the leading cause of inherited mental impairment and is associated with expansions of CGG repeats within the FMR1 gene. To detect expanded CGG repeats, we developed a dual-mode single-molecule fluorescence assay that allows acquisition of two parallel, independent measures of repeat number based on (1) the number of Cy3-labeled probes bound to the repeat region and (2) the physical length of the electric field-linearized repeat region, obtained from the relative position of a single Cy5 dye near the end of the repeat region. Using target strands derived from cell-line DNA with defined numbers of CGG repeats, we show that this assay can rapidly and simultaneously measure the repeats of a collection of individual sample strands within a single field of view. With a low occurrence of false positives, the assay differentiated normal CGG repeat lengths (CGG N , N = 23) and expanded CGG repeat lengths (CGG N , N = 118), representing a premutation disease state. Further, mixtures of these DNAs gave results that correlated with their relative populations. This strategy may be useful for identifying heterozygosity or for screening collections of individuals, and it is readily adaptable for screening other repeat disorders.


Fragile X syndrome Single molecule Total internal reflection fluorescence microscopy (TIRFM) Heterozygosity Dual mode Photobleaching Diagnostic assay 


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Brian Cannon
    • 1
  • Cynthia Pan
    • 1
  • Liangjing Chen
    • 2
  • Andrew G. Hadd
    • 2
  • Rick Russell
    • 1
    Email author
  1. 1.Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular BiologyThe University of TexasAustinUSA
  2. 2.Asuragen Inc.AustinUSA

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