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

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Abstract

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.

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References

  1. Brouwer, J. R., Willemsen, R., & Oostra, B. A. (2009). Microsatellite repeat instability and neurological disease. Bioessays, 1, 71–83.

    Article  Google Scholar 

  2. López Castel, A., Cleary, J. D., & Pearson, C. E. (2010). Repeat instability as the basis for human diseases and as a potential target for therapy. Nature Reviews Molecular Cell Biology, 3, 165–170.

    Article  Google Scholar 

  3. McMurray, C. T. (2010). Mechanisms of trinucleotide repeat instability during human development. Nature Reviews Genetics, 11, 786–799.

    Article  CAS  Google Scholar 

  4. Penagarikano, O., Mulle, J. G., & Warren, S. T. (2007). The pathophysiology of fragile X syndrome. Annual Review of Genomics and Human Genetics, 8, 109–129.

    Article  CAS  Google Scholar 

  5. Hagerman, R. J., Hull, C. E., Safanda, J. F., Carpenter, I., Staley, L. W., O’Connor, R. A., et al. (1994). High functioning fragile X males: Demonstration of an unmethylated fully expanded FMR-1 mutation associated with protein expression. American Journal of Medical Genetics, 4, 298–308.

    Article  Google Scholar 

  6. Coffey, S. M., Cook, K., Tartaglia, N., Tassone, F., Nguyen, D. V., Pan, R., et al. (2008). Expanded clinical phenotype of women with the FMR1 premutation. American Journal of Medical Genetics A, 146A, 1009–1016.

    Article  Google Scholar 

  7. Terracciano, A., Pomponi, M. G., Marino, G. M., Chiurazzi, P., Rinaldi, M. M., Dobosz, M., et al. (2004). Expansion to full mutation of a FMR1 intermediate allele over two generations. European Journal of Human Genetics, 12, 333–336.

    Article  CAS  Google Scholar 

  8. Fernandez-Carvajal, I., Lopez Posadas, B., Pan, R., Raske, C., Hagerman, P. J., & Tassone, F. (2009). Expansion of an FMR1 grey-zone allele to a full mutation in two generations. Journal of Molecular Diagnostics, 11, 306–310.

    Article  CAS  Google Scholar 

  9. Chen, L., Hadd, A., Sah, S., Filipovic-Sadic, S., Krosting, J., Sekinger, E., et al. (2010). An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis. Journal of Molecular Diagnostics, 12, 589–600.

    Article  CAS  Google Scholar 

  10. Lyon, E., Laver, T., Yu, P., Jama, M., Young, K., Zoccoli, M., et al. (2010). A simple, high-throughput assay for fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis. Journal of Molecular Diagnostics, 12, 505–511.

    Article  CAS  Google Scholar 

  11. Hantash, F. M., Goos, D. G., Tsao, D., Quan, F., Buller-Burckle, A., Peng, M., et al. (2010). Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: Implications for fragile X syndrome carrier and newborn screening. Genetics Medicine, 12, 162–173.

    Article  CAS  Google Scholar 

  12. Khaniani, M. S., Kalitsis, P., Burgess, T., & Slater, H. R. (2008). An improved diagnostic PCR assay for identification of cryptic heterozygosity for CGG triplet repeat alleles in the fragile X gene (FMR1). Molecular Cytogenetics, 1, 5.

    Article  Google Scholar 

  13. Todorov, T., Todorova, A., Georgieva, B., & Mitev, V. (2010). A unified rapid PCR method for detection of normal and expanded trinucleotide alleles of CAG repeats in huntington chorea and CGG repeats in fragile X syndrome. Molecular Biotechnology, 2, 150–154.

    Article  Google Scholar 

  14. Filipovic-Sadic, S., Sah, S., Chen, L., Krosting, J., Sekinger, E., Zhang, W., et al. (2010). A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome. Clinical Chemistry, 56, 399–408.

    CAS  Google Scholar 

  15. Pushkarev, D., Neff, N. F., & Quake, S. R. (2009). Single-molecule sequencing of an individual human genome. Nature Biotechnology, 27, 847–850.

    Article  CAS  Google Scholar 

  16. Eid, J., Fehr, A., Gray, J., Luong, K., Lyle, J., Otto, G., et al. (2009). Real-time DNA sequencing from single polymerase molecules. Science, 323, 133–138.

    Article  CAS  Google Scholar 

  17. Schlapak, R., Kinns, H., Wechselberger, C., Hesse, J., & Howorka, S. (2007). Sizing trinucleotide repeat sequences by single-molecule analysis of fluorescence brightness. ChemPhysChem, 8, 1618–1621.

    Article  CAS  Google Scholar 

  18. Jo, K., Dhingra, D. M., Odijk, T., de Pablo, J. J., Graham, M. D., Runnheim, R., et al. (2007). A single-molecule barcoding system using nanoslits for DNA analysis. Proceedings of the National Academy of Sciences USA, 8, 2673–2678.

    Article  Google Scholar 

  19. Xiao, M., Gordon, M. P., Phong, A., Ha, C., Chan, T. F., Cai, D., et al. (2007). Determination of haplotypes from single DNA molecules: A method for single-molecule barcoding. Human Mutation, 9, 913–921.

    Article  Google Scholar 

  20. Burton, R. E., White, E. J., Foss, T. R., Phillips, K. M., Meltzer, R. H., Kojanian, N., et al. (2010). A microfluidic chip-compatible bioassay based on single-molecule detection with high sensitivity and multiplexing. Lab on a Chip, 10, 843–851.

    Article  CAS  Google Scholar 

  21. Chan, E. Y., Goncalves, N. M., Haeusler, R. A., Hatch, A. J., Larson, J. W., Maletta, A. M., et al. (2004). DNA mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags. Genome Research, 14, 137–1146.

    Article  Google Scholar 

  22. Protozanova, E., Zhang, M., White, E. J., Mollova, E. T., Broeck, D. T., Fridrikh, S. V., et al. (2010). Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection. Analytical Biochemistry, 1, 83–90.

    Article  Google Scholar 

  23. Stoddart, D., Heron, A. J., Mikhailova, E., Maglia, G., & Bayley, H. (2009). Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proceedings of the National Academy of Sciences USA, 106, 7702–7707.

    Article  CAS  Google Scholar 

  24. Levine, P. M., Gong, P., Levicky, R., & Shepard, K. L. (2009). Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics. Biosensor Bioelectronics, 7, 1995–2001.

    Article  Google Scholar 

  25. Rothberg, J. M., Hinz, W., Rearick, T. M., Schultz, J., Mileski, W., Davey, M., et al. (2011). An integrated semiconductor device enabling non-optical genome sequencing. Nature, 475, 348–352.

    Article  CAS  Google Scholar 

  26. Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y. E., & Selvin, P. R. (2003). Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science, 300, 2061–2065.

    Article  CAS  Google Scholar 

  27. Qu, X., Wu, D., Mets, L., & Scherer, N. F. (2004). Nanometer-localized multiple single-molecule fluorescence microscopy. Proceedings of the National Academy of Sciences USA, 101, 11298–11303.

    Article  CAS  Google Scholar 

  28. Gordon, M. P., Ha, T., & Selvin, P. R. (2004). Single-molecule high-resolution imaging with photobleaching. Proceedings of the National Academy of Sciences USA, 101, 6462–6465.

    Article  CAS  Google Scholar 

  29. Churchman, L. S., Okten, Z., Rock, R. S., Dawson, J. F., & Spudich, J. A. (2005). Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proceedings of the National Academy of Sciences USA, 102, 1419–1423.

    Article  CAS  Google Scholar 

  30. Huang, B., Bates, M., & Zhuang, X. (2009). Super-resolution fluorescence microscopy. Annual Reviews in Biochemistry, 78, 993–1016.

    Article  CAS  Google Scholar 

  31. Nadel, Y., Weisman-Shomer, P., & Fry, M. (1995). The fragile X syndrome single strand d(CGG)n nucleotide repeats readily fold back to form unimolecular hairpin structures. Journal of Biological Chemistry, 270, 28970–28977.

    Article  CAS  Google Scholar 

  32. Paiva, A. M., & Sheardy, R. D. (2004). Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers. Biochemistry, 43, 14218–14227.

    Article  CAS  Google Scholar 

  33. Jarem, D. A., Huckaby, L. V., & Delaney, S. (2010). AGG interruptions in (CGG)(n) DNA repeat tracts modulate the structure and thermodynamics of non-B conformations in vitro. Biochemistry, 49, 6826–6837.

    Article  CAS  Google Scholar 

  34. Völker, J., Klump, H. H., & Breslauer, K. J. (2008). DNA energy landscapes via calorimetric detection of microstate ensembles of metastable macrostates and triplet repeat diseases. Proceedings of the National Academy of Sciences USA, 105, 18326–18330.

    Article  Google Scholar 

  35. Maier, B., Seifert, U., & Rädler, J. O. (2002). Elastic response of DNA to external electric fields in two dimensions. Europhysics Letters, 60, 622–628.

    Article  CAS  Google Scholar 

  36. Randall, G. C., Schultz, K. M., & Doyle, P. S. (2006). Methods to electrophoretically stretch DNA: Microcontractions, gels, and hybrid gel-microcontraction devices. Lab on a Chip, 6, 516–525.

    Article  CAS  Google Scholar 

  37. Nelson, P. C., Zurla, Z., Brogioli, D., Beausang, J. F., Finzi, L., & Dunlap, D. (2006). Tethered particle motion as a diagnostic of DNA tether length. Journal of Physical Chemistry B, 110, 17260–17267.

    Article  CAS  Google Scholar 

  38. American College of Obstetricians and Gynecologists. (2010). Carrier screening for fragile X syndrome. Committee Opinion No. 469. Obstetrics and Gynecology, 116, 1008–1010.

    Article  Google Scholar 

  39. Spector, E. B., & Kronquist, K. E. (2005) Fragile X: Technical standards and guidelines. ACMG standards and guidelines for clinical genetics laboratories.

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Correspondence to Rick Russell.

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Cannon, B., Pan, C., Chen, L. et al. A Dual-Mode Single-Molecule Fluorescence Assay for the Detection of Expanded CGG Repeats in Fragile X Syndrome. Mol Biotechnol 53, 19–28 (2013). https://doi.org/10.1007/s12033-012-9505-z

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  • DOI: https://doi.org/10.1007/s12033-012-9505-z

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