Abstract
The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources 1,2. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides 3,4,5, for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Wiegant, J. et al. An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization. J. Histochem. Cytochem. 44, 525–529 (1996).
Tanke, H.J. et al. New strategy for multi-colour fluorescence in situ hybridization: COBRA: COmbined Binary RAtio labelling. Eur. J. Hum. Genet. 7, 2–11 (1999).
Langer, P.R., Waldrop, A.A. & Ward, D.C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U S A 78, 6633–6637 (1981).
Shimkus, M.L., Guaglianone, P. & Herman, T.M. Synthesis and characterization of biotin-labeled nucleotide analogs. DNA 5, 247–255 (1986).
Cook, A.F., Vuocolo, E. & Brakel, C.L. Synthesis and hybridization of a series of biotinylated oligonucleotides. Nucleic Acids Res. 16, 4077–4095 (1988).
Speicher, M.R., Ballard, S.G. & Ward, D.C. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nature Genet. 12, 368–375 (1996).
Schrock, E. et al. Multicolor spectral karyotyping of human chromosomes. Science 273, 494–497 (1996).
Henegariu, O., Heerema, N.A., Bray-Ward, P. & Ward, D.C. Colour-changing karyotyping: an alternative to M-FISH/SKY. Nature Genet. 23, 263–264 (1999).
Telenius, H. et al. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13, 718–725 (1992).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Henegariu, O., Bray-Ward, P. & Ward, D. Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling. Nat Biotechnol 18, 345–348 (2000). https://doi.org/10.1038/73815
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/73815
This article is cited by
-
A protein assembly mediates Xist localization and gene silencing
Nature (2020)
-
High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa
BMC Genetics (2015)
-
Remodeling of Three-Dimensional Organization of the Nucleus during Terminal Keratinocyte Differentiation in the Epidermis
Journal of Investigative Dermatology (2013)
-
Genomic rearrangements at the FRA2H common fragile site frequently involve non-homologous recombination events across LTR and L1(LINE) repeats
Human Genetics (2012)
-
Fetal derived embryonic-like stem cells improve healing in a large animal flexor tendonitis model
Stem Cell Research & Therapy (2011)
