Abstract:Nucleoside triphosphates I with 3'-O-blocking groups that are both photolabile and fluorescent were required to investigate the viability of a strategy for sequencing DNA in a combinatorial fashion (see Figure 1). Four compounds were prepared to realize this goal. Two of them, 14 a and 14 t, had dansyl-functionalized, 3'-O-(2''-nitrobenzyl) ether groups, while the other two, 18 a and 18 t, had similar pendant carbonate groups. Tests for incorporation of these analogues were performed by using five different DN… Show more
“…T7 Sequenase v2.0 and Klenow (exo-)] have shown that this thymidine carbonate is not incorporated by these DNA replicating enzymes. The unsuccessful DNA polymerase-mediated incorporation of 3′- O -modified nucleotides has also been reported by the Burgess group (37) and was explained as follows: the 3′ position on the deoxyribose is very close to the amino acid residues in the active site of the polymerase, and the polymerase is therefore sensitive to modification in this area of the ribose ring, especially with a bulky fluorophore such as rhodamine derivatives. Figure 1.Structures of cleavable fluorescent dNTPs 1 and heterobifunctional cross-linking reagent Pydec-Im.…”
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® 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.
“…T7 Sequenase v2.0 and Klenow (exo-)] have shown that this thymidine carbonate is not incorporated by these DNA replicating enzymes. The unsuccessful DNA polymerase-mediated incorporation of 3′- O -modified nucleotides has also been reported by the Burgess group (37) and was explained as follows: the 3′ position on the deoxyribose is very close to the amino acid residues in the active site of the polymerase, and the polymerase is therefore sensitive to modification in this area of the ribose ring, especially with a bulky fluorophore such as rhodamine derivatives. Figure 1.Structures of cleavable fluorescent dNTPs 1 and heterobifunctional cross-linking reagent Pydec-Im.…”
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® 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.
“…terization by MALDI-TOF MS. 3Ј-O-modified nucleotides pose a great challenge for incorporation by natural polymerase, especially when the 3Ј-O-labeling group is a bulky one (31,32). To verify that the NRTs can be recognized by polymerase as substrates in a polymerase reaction, we performed extension reactions with four different primers corresponding to different regions of a DNA template whose next complementary base was either A, C, G, or T. A 9°N polymerase (exo-)A485L/Y409V, which has been shown previously to incorporate the 3Ј-Omodified nucleotides (21,23), was used in the polymerase extension reaction.…”
Section: Polymerase Extension Using 3-o-modified Nucleotides and Charac-mentioning
Pyrosequencing is a method used to sequence DNA by detecting the pyrophosphate (PPi) group that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction. However, this method has an inherent difficulty in accurately deciphering the homopolymeric regions of the DNA templates. We report here the development of a method to solve this problem by using nucleotide reversible terminators. These nucleotide analogues are modified with a reversible chemical moiety capping the 3-OH group to temporarily terminate the polymerase reaction. In this way, only one nucleotide is incorporated into the growing DNA strand even in homopolymeric regions. After detection of the PPi for sequence determination, the 3-OH of the primer extension products is regenerated through different deprotection methods. Using an allyl or a 2-nitrobenzyl group as the reversible moiety to cap the 3-OH of the four nucleotides, we have synthesized two sets of 3-O-modified nucleotides, 3-O-allyl-dNTPs and 3-O-(2-nitrobenzyl)-dNTPs as reversible terminators for pyrosequencing. The capping moiety on the 3-OH of the DNA extension product is efficiently removed after PPi detection by either a chemical method or photolysis. To sequence DNA, templates containing homopolymeric regions are immobilized on Sepharose beads, and then extension-signal detection-deprotection cycles are conducted by using the nucleotide reversible terminators on the DNA beads to unambiguously decipher the sequence of DNA templates. Our results establish that this reversible-terminator-pyrosequencing approach can be potentially developed into a powerful methodology to accurately determine DNA sequences.nucleotide reversible terminator ͉ sequencing by synthesis D NA sequencing is a fundamental tool for biological science. The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. To overcome the limitations of the current electrophoresisbased sequencing technology (2-5), a variety of new DNAsequencing methods have been investigated with an aim to eventually realize the goal of the $1,000 genome. Such approaches include sequencing by hybridization (6), mass spectrometry-based sequencing (7-9), sequence-specific detection of DNA using engineered nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis approaches such as pyrosequencing (12), sequencing of single DNA molecules (13,14), and polymerase colonies (15) have been widely explored.Pyrosequencing is a method to sequence DNA by detecting the pyrophosphate (PPi) that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction (12). In this approach, each of the four nucleotides is added sequentially with a mixture of enzymes and substrates in addition to the usual polymerase reactio...
“…Within the scope of this study, several 3′-blocking groups were investigated, including bulky esters[ 7 ] and ethers[ 8 ] with the label attached to the blocking group and small groups. Some examples are the 3′- O -(2-nitrobenzyl) group investigated by Metzker and co-workers[ 6 ] and Welch and Burgess,[ 8 , 11 ] the 3′- O -allyl group reported by Metzker,[ 6 ] Ju,[ 12 ] and Kim,[ 13 ] or the 3′- O -azidomethyl group, which was used by Ju and co-workers[ 14 , 15 ] and was also realised in a commercially available device, the Genome Analyzer developed by Illumina/Solexa. [ 16 , 17 ] Other interesting groups are the 3′- O -NH 2 group from Benner and co-workers,[ 18 ] the 3′- O -(2-cyanoethoxy)methyl group reported by us,[ 19 ] or some 3′-blocking groups removable under mild reducing or mild acidic conditions reported by Kwiatkowski.…”
Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.
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