Modified Synthesis of 6-carboxyfluorescein (6-FAM): Application to Probe Labeling for Conventional Cytogenetics
Alexander Stepakov
Department of Organic Chemistry, Institute of Chemistry, Saint-Petersburg State University, Universitetskii Prosp., 26, Saint-Petersburg, 198504, Russian Federation
Svetlana Galkina
Department of Cytology and Histology, Biological Faculty, Saint-Petersburg State University, Oranienbaumskoye Shosse, 2, Saint-Petersburg, 198504, Russian Federation.
Denis Bogomaz
Department of Genetics and Biotechnology, Biological Faculty, Saint-Petersburg State University, Botanicheskaya ul., 17, Saint-Petersburg, 198504, Russian Federation
Elena Gaginskaya
Department of Cytology and Histology, Biological Faculty, Saint-Petersburg State University, Oranienbaumskoye Shosse, 2, Saint-Petersburg, 198504, Russian Federation
Alsu Saifitdinova *
Department of Cytology and Histology, Biological Faculty, Saint-Petersburg State University, Oranienbaumskoye Shosse, 2, Saint-Petersburg, 198504, Russian Federation.
*Author to whom correspondence should be addressed.
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.
Keywords: 6-carboxyfluorescein phosphoramidite, p-nitrophenyl ether of carboxyfluorescein, fluorescent in situ hybridization, fluorochrome, oligonucleotide synthesis, FISH probe.