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Research Article Open access CC BY 4.0

Modified Synthesis of 6-carboxyfluorescein (6-FAM): Application to Probe Labeling for Conventional Cytogenetics

Alexander Stepakov, Svetlana Galkina, Denis Bogomaz, Elena Gaginskaya, Alsu Saifitdinova

Current Journal of Applied Science and Technology · pp. 423–428 · Published 24 Feb 2015

10.9734/BJAST/2015/15991

Abstract

Aims: Fluorescent in situ hybridization (FISH), the routine technique of molecular cytogenetics, is widely used to detect and localize the presence of specific nucleic acids sequences in chromosomes, cell nucleus space, cells and tissue samples through the use of highly complementary probes to targets sequence. Expansion of FISH method application for research purposes and medical diagnostics requires efficient and low-cost production of labeled nucleic acid probes. Methodology and Results: We developed an effective method of fluorescein hydroxyalkyl carboxamides synthesis. This modification of the basic protocol of 6-carboxyfluorescein (6-FAM) synthesis enabled the production of highly reactive conjugate perfectly suitable for terminal labeling of newly generated oligonucleotides. Efficiency of 6-FAM labeled oligonucleotides obtained by the use of modified protocol has been proved for conventional cytogenetics. Conclusion: The suggested procedure of 6-FAM labeled oligonucleotides synthesis allows obtaining the high yield of directly labeled FISH probes. The introduction of this method into practice of cytogenetic studies will improve their efficiency and reduce the cost of an examination.

6-carboxyfluorescein phosphoramidite p-nitrophenyl ether of carboxyfluorescein fluorescent in situ hybridization fluorochrome oligonucleotide synthesis FISH probe.

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