A New Method of Synthesis of Fluorescently Labelled Oligonucleotides and Their Application in DNA Sequencing

Markiewicz, Wojciech T.; Gröger, G.; Rösch, Rudi; Zebrowska, Anna; Seliger, H.

doi:10.1080/07328319208017817

Cited by 16 publications

(10 citation statements)

References 44 publications

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“…Then we used DMAP as an additive, instead of HOBT, to improve coupling performance. A catalytic amount of DMAP is commonly used with carbodiimide coupling reagents (22)(23)(24)(25)(26)(27), and recently, we found that DMAP greatly increases the speed and efficiency of amide formation with uronium or phosphonium coupling reagents (2). However, there have only been a few other reports of DMAP used with uronium (47-50) or phos-phonium (46,51) coupling reagents.…”

Section: Resultsmentioning

confidence: 99%

“…This problem becomes even more pronounced when coupling must be made through a secondary alcohol (22,23) such as the 3′-OH group in nucleosides. Therefore, we wanted to find coupling conditions which would form the required ester linkages faster and more efficiently than the carbodiimide coupling reagents previously used (22)(23)(24)(25)(26)(27).…”

Section: Introductionmentioning

confidence: 99%

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Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents

Pon

Sanghvi

1999

Bioconjugate Chem.

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Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 micromol/g) and rapid coupling reactions (< or = 10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3'-succinyl or 3'-hydroquinone-O, O'-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3'-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents

Pon

Sanghvi

1999

Bioconjugate Chem.

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“…nucleic acids and synthetic oligonucleotides, are extremely useful in many areas of molecular biology. The automated DNA sequencing method utilizes rhodamine derivatives (11)(12)(13) and fluorescein derivatives (14)(15)(16)(17)(18)(19). Other non-radioactive systems [fluorochromes (20)(21)(22)(23), enzymes (24)(25)(26)(27), biotin based (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) or hapten (39)(40)(41)(42), digoxigenin (43 -44)] are used to label oligonucleotide probes for diagnostic applications.…”

Section: Introductionmentioning

confidence: 99%

1,N⁶-etheno deoxy and ribo adenoGine and 3,N⁴-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules

Srivastava¹,

Raza²,

Misra³

1994

Nucl Acids Res

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Synthesis of 1,N6-etheno-2'-deoxyadenosine, 3,N4-etheno-2'-deoxycytidine, and further chemistry on both deoxy and ribo series etheno nucleosides produces the corresponding phosphoramidites. These novel phosphoramidites are introduced selectively, quantitatively, and at specific positions at single or multiple sites into DNA or RNA sequences. The purification and chemistry involved in the synthesis of these products has been optimized to achieve the purity in excess of 99%. The resulting phosphoramidites were tested for their ability to couple and produce poly deoxy and ribonucleotides by solid phase chemistry. The coupling efficiency achieved was greater than 99% per step. Due to the instability of these etheno compounds in acidic and basic medium, various criteria to obtain pure oligomers have been established. The selective introduction of these fluorescent nucleosides into defined sequence DNA and RNA molecule will greatly facilitate the structure-function studies of various RNAs, protein-RNA structures, and DNA-RNA based diagnostics applications. The characteristic and high fluorescent intensity (detection below 1 x 10(-9) M for adenosine sites and below 1 x 10(-7) M for cytidine sites) is particularly suited for the biochemical and biological research and product development applications. The usefulness of these etheno containing modified sequences as sequencing and amplification primers is demonstrated by their full participation in polymerase chain reaction experiments.

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“…Initially limited to the commercially available uridine derivative, the procedure was subsequently extended to fluorescein-ATP and -CTP (Igloi, 1996a)(essential for retaining the complementarity of the 3' terminal nucleotide) and remains to date the only direct enzymatic, template independent method for labelling preexisting primers. Despite the proven advantages of this enzymatic procedure in terms of speed and economy during a genome project , Markiewicz et al (1997), apparently unaware of previous publications, described the chemical synthesis of 3' end labelled primers and their application in DNA sequencing.…”

Section: Reaction According To Sangermentioning

confidence: 99%

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

Igloi¹

1998

Electron. J. Biotechnol.

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Rapid, large-scale genome as well as diagnostic DNA sequencing projects are, at present, dependent on the use of sensitive automated sequencers that rely on the detection of fluorescent signals. This emission is not an intrinsic property of the biomolecules but is a property of an optical label that must be incorporated before, during or after the sequence specific reaction. The choice of strategy, that is to be reviewed here, is a function of both the chemistry and the enzymology of the system as well as the nature of the fractionation and the physical parameters of the detection. One may differentiate beween the labelling of the primer, incorporation of the label during elongation or base specific termination with labelled terminators. In reviewing the development of each of these strategies, the conclusion is reached that, whereas labelled primers have universal applicability, the current generation of fluorescent terminators have in, conjunction with appropriate DNA polymerases, attained a refinement that strongly favours their use. However, since they are not available for all commercial sequencing systems, the alternative procedures are not merely of historic interest but are an essential component in DNA sequencing protocols. The possibility of automated direct sequence analysis of RNA has not been ignored completely.

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A New Method of Synthesis of Fluorescently Labelled Oligonucleotides and Their Application in DNA Sequencing

Abstract: A new approach to the chemical synthesis of oligonucleotides bearing reporter functional groups based on the modification of cytosine residues during the final deprotection step is reported. The application of the fluorescently labelled primer for the automated DNA sequencing is shown.

Cited by 16 publications

References 44 publications

Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents

Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents

1,N⁶-etheno deoxy and ribo adenoGine and 3,N⁴-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

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A New Method of Synthesis of Fluorescently Labelled Oligonucleotides and Their Application in DNA Sequencing

Abstract: A new approach to the chemical synthesis of oligonucleotides bearing reporter functional groups based on the modification of cytosine residues during the final deprotection step is reported. The application of the fluorescently labelled primer for the automated DNA sequencing is shown.

Cited by 16 publications

References 44 publications

Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents

Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents

1,N6-etheno deoxy and ribo adenoGine and 3,N4-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

Contact Info

Product

Resources

About

1,N⁶-etheno deoxy and ribo adenoGine and 3,N⁴-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules