Limits...
A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis.

Turcatti G, Romieu A, Fedurco M, Tairi AP - Nucleic Acids Res. (2008)

Bottom Line: In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base.Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out).Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

View Article: PubMed Central - PubMed

Affiliation: Manteia Predictive Medicine S.A., Zone Industrielle, Coinsins, CH-1267, Switzerland. gerardo.turcatti@epfl.ch

ABSTRACT
Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

Show MeSH
Structures of dNTP(AP3)-SS-AF594.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275100&req=5

Figure 2: Structures of dNTP(AP3)-SS-AF594.

Mentions: We thus focused on the design and synthesis of nucleotide analogs, in which the fluorophore is linked to the pyrimidine or purine bases through a cleavable disulfide linker. Indeed, it is known that most of DNA polymerases are highly tolerable to extensive modifications with large groups such as fluorescent dyes at the 5 position of pyrimidines (C and T) and 7 position of purines (A and G) (38,39). Furthermore, we postulated that the steric hindrance of the cleavable fluorophore may confer some terminating properties to these free 3′-OH-modified nucleotides. Recently, the use of 3′-OH-free fluorescent deoxynucleotides as terminators of DNA polymerization has been reported for single bases extensions in microarrays (40) supporting the fact that 3′-OH-modified nucleotides can act as terminators for DNA polymerase-mediated elongation under certain experimental conditions. It is clear that for sequencing applications the removal of the label is needed and in this context, our goal was to prepare the four chemically cleavable fluorescent nucleotide analogs 2–5, with an Alexa Fluor® 594 dye attached to the 5 (or 7) position of pyrimidine (or purine) base via a cleavable disulfide linker (Figure 2). Alexa Fluor® 594 (AF594) is a water-soluble analogue of Texas Red® (TR) fluorophore; it was chosen to avoid fluorescent background causing by the non-specific adsorption of fluorescent species (e.g. fluorescent nucleotides and/or free dye) over DNA biochip surface after the incorporation and cleavage steps of binary sequencing cycle. Such phenomenon was observed with Texas Red® despite of adding further washing steps to the SBS scheme. In addition to their excellent water-solubility, (1) the wide variety of Alexa Fluor® dyes spanning the visible and near-infrared spectrum would allow us to develop a series of different fluorescent nucleotides for the design of a four-color DNA-sequencing approach, and (2) they exhibit excellent emission intensity and photostability when conjugated to a biomolecule such as DNA (41,42).Figure 2.


A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis.

Turcatti G, Romieu A, Fedurco M, Tairi AP - Nucleic Acids Res. (2008)

Structures of dNTP(AP3)-SS-AF594.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275100&req=5

Figure 2: Structures of dNTP(AP3)-SS-AF594.
Mentions: We thus focused on the design and synthesis of nucleotide analogs, in which the fluorophore is linked to the pyrimidine or purine bases through a cleavable disulfide linker. Indeed, it is known that most of DNA polymerases are highly tolerable to extensive modifications with large groups such as fluorescent dyes at the 5 position of pyrimidines (C and T) and 7 position of purines (A and G) (38,39). Furthermore, we postulated that the steric hindrance of the cleavable fluorophore may confer some terminating properties to these free 3′-OH-modified nucleotides. Recently, the use of 3′-OH-free fluorescent deoxynucleotides as terminators of DNA polymerization has been reported for single bases extensions in microarrays (40) supporting the fact that 3′-OH-modified nucleotides can act as terminators for DNA polymerase-mediated elongation under certain experimental conditions. It is clear that for sequencing applications the removal of the label is needed and in this context, our goal was to prepare the four chemically cleavable fluorescent nucleotide analogs 2–5, with an Alexa Fluor® 594 dye attached to the 5 (or 7) position of pyrimidine (or purine) base via a cleavable disulfide linker (Figure 2). Alexa Fluor® 594 (AF594) is a water-soluble analogue of Texas Red® (TR) fluorophore; it was chosen to avoid fluorescent background causing by the non-specific adsorption of fluorescent species (e.g. fluorescent nucleotides and/or free dye) over DNA biochip surface after the incorporation and cleavage steps of binary sequencing cycle. Such phenomenon was observed with Texas Red® despite of adding further washing steps to the SBS scheme. In addition to their excellent water-solubility, (1) the wide variety of Alexa Fluor® dyes spanning the visible and near-infrared spectrum would allow us to develop a series of different fluorescent nucleotides for the design of a four-color DNA-sequencing approach, and (2) they exhibit excellent emission intensity and photostability when conjugated to a biomolecule such as DNA (41,42).Figure 2.

Bottom Line: In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base.Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out).Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

View Article: PubMed Central - PubMed

Affiliation: Manteia Predictive Medicine S.A., Zone Industrielle, Coinsins, CH-1267, Switzerland. gerardo.turcatti@epfl.ch

ABSTRACT
Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

Show MeSH