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Preparation of the fluorescein reagent for solid-phase oligonucleotide 5\'-labelling and its use for the synthesis of fluorescently labelled PCR primers for HIV-1 detection
| Content Provider | Semantic Scholar |
|---|---|
| Author | Dubey, I. Ya. Fedoryak, Olesya D. Tkachak, O. M. Fedoryak, D. M. |
| Copyright Year | 1995 |
| Abstract | Introduction. The progress in our understanding of gene structure and function has depended upon methods for detecting small amounts of nuc leic acids. The screening, isolation, structural and functional analysis of genes all involve the use and detection of labelled oligonucleotides. Ra dioactive labels are still very popular because they are easy to handle and provide a great sensitivity of detection. But an increasing number of highly sensitive non-radioactive bioanalytical systems are being developed to reduce or eliminate the problems associated with radioactivity [1, 2]. Non-isotopic reporter groups, in contrast to isotopic labels, are usually stable and have safety advantages. Fluorescent labelling is now widely used since fluorescent dyes are detected directly and at high sensitivity [3]. The latter is adequate for most applications in the nucleic acids stu dies except those which require ultimate sensitivity [1]. Oligonucleotides carrying fluorescent reporter groups are used as hybridization probes for the detection of specific nucleic acids, including diagnostic procedures in medicine [1—5], as primers for automated sequencing [6], in fluores cence microscopy [7], etc. We have recently started the studies of the fluorescent DNA labelling, particularly for AIDS detection. Polymerase chain reaction (PCR) is now used as diagnostic procedure in screening for HIV sequences [4]. It would be convenient for clinical laboratories to detect amplified fragments using a fluorescently labelled primers or probes. Here we describe the preparation of the reagent for solid-phase oligonucleotide labelling with fluorescein and its use for the synthesis of labelled primers for HIV-1 detection. Materials and Methods. 4,4-Dimethoxytrityl chloride (DMTrCl) and 1,2,4-trtazole (Tri) were purchased from Fluka (Switzerland), acrylamide and N,N'-methylene bisacrylamide for electrophoresis from Reanal (Hun gary). Another reagents and solvents were of home production. Solvents were dried according to [8]. Silicagel 60 F254 plates (Merck, Germany) were used for TLC analysis in the following solvent systems: CHC13СНзОН 9 : 1 (A), CHCI3 (B), iso-PrOH-conc. NH3-H20 7 : 2 : 1 (C). Trimethylsilyl silica (TMS-silica) was prepared as described [9]. Reversephase HPLC was performed on Pharmacia FPLC System (Sweden) using HR 5/2 column in the gradient of CH3CN in 0,1 M triethylammonium ace tate (pH 7.5) with elution rate 0,5 ml/min. Absorbance spectra were re corded on Specord UV-VIS spectrometer (Karl Zeiss Jena, Germany), fluorescence spectra were obtained on Hitachi MPF-4 spectrofluorimeter (Japan). Victoria-6M gene synthesizer (Novosibirsk, Russia) was used |
| Starting Page | 35 |
| Ending Page | 41 |
| Page Count | 7 |
| File Format | PDF HTM / HTML |
| DOI | 10.7124/bc.0003EB |
| Volume Number | 11 |
| Alternate Webpage(s) | http://www.biopolymers.org.ua/pdf/en/11/3/035/biopolym.cell-1995-11-3-035-en.pdf |
| Alternate Webpage(s) | https://doi.org/10.7124/bc.0003EB |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
