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Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.

ABSTRACT: DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.

SUBMITTER: Ju J

PROVIDER: S-EPMC1702316 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.

Project description:DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.

| S-EPMC2442126 | biostudies-literature

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

Project description: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.

| S-EPMC2275100 | biostudies-literature

DNA sequencing by synthesis using 3'-O-azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection.

Project description:As an alternative to fluorescence-based DNA sequencing by synthesis (SBS), we report here an approach using an azido moiety (N3) that has an intense, narrow and unique Raman shift at 2125 cm-1, where virtually all biological molecules are transparent, as a label for SBS. We first demonstrated that the four 3'-O-azidomethyl nucleotide reversible terminators (3'-O-azidomethyl-dNTPs) displayed surface enhanced Raman scattering (SERS) at 2125 cm-1. Using these 4 nucleotide analogues as substrates, we then performed a complete 4-step SBS reaction. We used SERS to monitor the appearance of the azide-specific Raman peak at 2125 cm-1 as a result of polymerase extension by a single 3'-O-azidomethyl-dNTP into the growing DNA strand and disappearance of this Raman peak with cleavage of the azido label to permit the next nucleotide incorporation, thereby continuously determining the DNA sequence. Due to the small size of the azido label, the 3'-O-azidomethyl-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for the azido moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS.

| S-EPMC4226440 | biostudies-literature

Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides.

Project description:We report four-color DNA sequencing by synthesis (SBS) on a chip, using four photocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) (PC, photocleavable; Bodipy, 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solution-phase polymerase reaction and the fluorophore could be cleaved using laser irradiation ( approximately 355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azido-labeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length.

| S-EPMC1087949 | biostudies-literature

Fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension.

Project description: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.

| S-EPMC3110862 | biostudies-other

Design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis.

Project description:DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3'-OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3'-OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3'-OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3'-modified photocleavable fluorescent nucleotide, 3'-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3'-OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS.

| S-EPMC1087948 | biostudies-literature

Improved DNA sequencing accuracy and detection of heterozygous alleles using manganese citrate and different fluorescent dye terminators.

Project description:The use of dideoxynucleotide triphosphates labeled with different fluorescent dyes (dye terminators) is the most versatile method for automated DNA sequencing. However, variation in peak heights reduces base-calling accuracy and limits heterozygous allele detection, favoring use of dye-labeled primers for this purpose. We have discovered that the addition of a manganese salt to the PE Applied Biosystems dye-terminator sequencing kits overcomes these limitations for the older rhodamine dyes as well as the more recent dichloro-rhodamine dyes (dRhodamine and BigDyes). Addition of manganese to reactions containing dRhodamine-based dye terminators produced the highest base-calling accuracy. This combination resulted in the most uniform electropherogram profiles, superior to those produced by BigDye terminators and published for dye primers, and facilitated detection of heterozygous alleles.

| S-EPMC310770 | biostudies-literature

Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection.

Project description:Any system, natural or human-made, is better understood if we analyze both its history and its structure. Here we combine structural analyses with a "Reconstructed Evolutionary Adaptive Path" (REAP) analysis that used the evolutionary and functional history of DNA polymerases to replace amino acids to enable polymerases to accept a new class of triphosphate substrates, those having their 3'-OH ends blocked as a 3(')-ONH(2) group (dNTP-ONH(2)). Analogous to widely used 2',3'-dideoxynucleoside triphosphates (ddNTPs), dNTP-ONH(2)s terminate primer extension. Unlike ddNTPs, however, primer extension can be resumed by cleaving an O-N bond to restore an -OH group to the 3'-end of the primer. REAP combined with crystallographic analyses identified 35 sites where replacements might improve the ability of Taq to accept dNTP-ONH(2)s. A library of 93 Taq variants, each having replacements at three or four of these sites, held eight variants having improved ability to accept dNTP-ONH(2) substrates. Two of these (A597T, L616A, F667Y, E745H, and E520G, K540I, L616A) performed notably well. The second variant incorporated both dNTP-ONH(2)sand ddNTPs faithfully and efficiently, supporting extension-cleavage-extension cycles applicable in parallel sequencing and in SNP detection through competition between reversible and irreversible terminators. Dissecting these results showed that one replacement (L616A), not previously identified, allows Taq to incorporate both reversible and irreversible terminators. Modeling showed how L616A might open space behind Phe-667, allowing it to move to accommodate the larger 3'-substituent. This work provides polymerases for DNA analyses and shows how evolutionary analyses help explore relationships between structure and function in proteins.

| S-EPMC2804741 | biostudies-literature

3'-O-modified nucleotides as reversible terminators for pyrosequencing.

Project description: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.

| S-EPMC2034218 | biostudies-literature

Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates.

Project description:We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the ?-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-?-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.

| S-EPMC3064798 | biostudies-other

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