Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing

A novel series of charge-modified, dye-labeled 2′,3′-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase™ II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.     

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates

Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2′-deoxyribonucleoside-5′-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5′-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In addition, we attached novel fluorophore derivatives. The labeled products were obtained in at least 96% purity after HPLC purification. Enzymatic incorporation into DNA and subsequent extension of the modified DNA chain were studied. Vent^R exo^– DNA polymerase and a defined template–primer system were used to analyze each dye-labeled dNTP derivative. Our data suggest that the incorporation efficiency depends on the selected dye, the nucleobase or a combination of both.     

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50°C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.     

New dye-labeled terminators for improved DNA sequencing patterns.

We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.     

Novel cyanine dye-labeled dideoxynucleoside triphosphates for DNA sequencing

Single color cyanine dye-labeled (Cy 5.0 and Cy 5.5) dideoxynucleoside-5'-triphosphates, or 'terminators', containing different spacer lengths were synthesized and evaluated for efficacy in DNA sequencing methods using a modified thermally stable DNA polymerase. The single color cyanine dye terminators were formulated into two separate sets of sequencing mixes, one for Cy 5.0 and the other for Cy 5.5, and evaluated on different automated sequencing platforms. Each set of mixes included two pyrimidine terminators with 17-atom linkers and two purine terminators with 10-atom linkers between the dye and the nucleotide. The two sets of cyanine dye-labeled terminators chosen for this cycle sequencing study produced improved band patterns with band uniformity similar to that obtained with dye-primer sequencing methods.     

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides

Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5′-amino-5′-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5′-amino-2′,5′-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5′-azido-2′,5′-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5′-amino-5′-deoxythymidine to the corresponding 5′-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension–cleavage procedure in genomic sequence analysis is discussed.     

Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms

Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3′-N5′ phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5′-amino-5′-deoxynucleoside 5′-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate. Utilizing this approach, a polymorphic site in the human ADRB3 gene was interrogated. Primer extensions with phosphoramidate analogs of dNTPs allowed for unambiguous discrimination of all possible genotypes.     

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3′→5′ exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.     

Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.     

Selective Inhibition of DNA Chain Elongation Catalyzed by DNA Polymerases

Abstract

The results of series of works on the properties of a large number of nucleoside 5′-triphosphates analogs in the reaction catalyzed by several DNA polymerases are summarized.

Molecular mechanisms of substrate selection by DNA polymerases are not studied in detail. Therefore we have undertaken a comparative analysis of DNA polymerases from different sources employing nucleoside 5′-triphosphate analogs capable of incorporating into DNA chains terminating these chains elongation. Synthesis of a large line of nucleoside 5′-triphosphate analogs with substitution at the sugar residue has been performed. DNA polymerases have been isolated, and the synthesis of DNA has been studied using phage M13 DNA or phage MS2 RNA with synthetic deoxyoligonucleoti-de primers. The molecular mechanism of the substrate action has been determined by PAG electrophoresis of the reaction     

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3′-unpaired overhang, a 5′ overhang, or both 3′ and 5′ overhangs. The sequence of the 5′ overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3′ strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5′ strand, but since such molecules cannot be processed into nicked intermediates, the 3′-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5′ overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.     

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms

Different chemical methods used to attach oligonucleotides by their 5′-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5′-specific linkage that satisfyingly withstands PCR conditions and leaves the 3′-ends available for DNA polymerase activity. The best results were obtained with 5′-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5′-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.     

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.     

Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus

Reaction intermediates formed during the degradation of linear PM2, T5, and λ DNA by herpes simplex virus (HSV) DNase have been examined by agarose gel electrophoresis. Digestion of T5 DNA by HSV type 2 (HSV-2) DNase in the presence of Mn²⁺ (endonuclease only) gave rise to 6 major and 12 minor fragments. Some of the fragments produced correspond to those observed after cleavage of T5 DNA by the single-strand-specific S1 nuclease, indicating that the HSV DNase rapidly cleaves opposite a nick or gap in a duplex DNA molecule. In contrast, HSV DNase did not produce distinct fragments upon digestion of linear PM2 or λ DNA, which do not contain nicks. In the presence of Mg²⁺, when both endonuclease and exonuclease activities of the HSV DNase occur, most of the same distinct fragments from digestion of T5 DNA were observed. However, these fragments were then further degraded preferentially from the ends, presumably by the action of the exonuclease activity. Unit-length λ DNA, EcoRI restriction fragments of λ DNA, and linear PM2 DNA were also degraded from the ends by HSV DNase in the same manner. Previous studies have suggested that the HSV exonuclease degrades in the 3′ → 5′ direction. If this is correct, and since only 5′-monophosphate nucleosides are produced, then HSV DNase should “activate” DNA for DNA polymerase. However, unlike pancreatic DNase I, neither HSV-1 nor HSV-2 DNase, in the presence of Mg²⁺ or Mn²⁺, activated calf thymus DNA for HSV DNA polymerase. This suggests that HSV DNase degrades both strands of a linear double-stranded DNA molecule from the same end at about the same rate. That is, HSV DNase is apparently capable of degrading DNA strands in the 3′ → 5′ direction as well as in the 5′ → 3′ direction, yielding progressively smaller double-stranded molecules with flush ends. Except with minor differences, HSV-1 and HSV-2 DNases act in a similar manner.     

Ligase-mediated construction of branched DNA strands: a novel DNA joining activity catalyzed by T4 DNA ligase

Branched nucleic acid strands exist as intermediates in certain biological reactions, and bifurcating DNA also presents interesting opportunities in biotechnological applications. We describe here how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5′ end but two distinct 3′ ends that extend from the 2′ and 3′ carbons, respectively, of an internal nucleotide. The nature of the reaction products is investigated, and optimal reaction conditions are reported for the construction of branched oligonucleotides. We discuss the utility of these branched DNA nanostructures for gene detection.     

Synthesis and Energy Transfer Efficiency of FRET Terminators Derived from Different Linkers

Abstract

A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5′-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.     

Electron attachment-induced DNA single-strand breaks at the pyrimidine sites

To elucidate the contribution of pyrimidine in DNA strand breaks caused by low-energy electrons (LEEs), theoretical investigations of the LEE attachment-induced C_3′–O_3′, and C_5′–O_5′ σ bond as well as N-glycosidic bond breaking of 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate were performed using the B3LYP/DZP++ approach. The base-centered radical anions are electronically stable enough to assure that either the C–O or glycosidic bond breaking processes might compete with the electron detachment and yield corresponding radical fragments and anions. In the gas phase, the computed glycosidic bond breaking activation energy (24.1 kcal/mol) excludes the base release pathway. The low-energy barrier for the C_3′–O_3′ σ bond cleavage process (∼6.0 kcal/mol for both cytidine and thymidine) suggests that this reaction pathway is the most favorable one as compared to other possible pathways. On the other hand, the relatively low activation energy barrier (∼14 kcal/mol) for the C_5′–O_5′ σ bond cleavage process indicates that this bond breaking pathway could be possible, especially when the incident electrons have relatively high energy (a few electronvolts). The presence of the polarizable medium greatly increases the activation energies of either C–O σ bond cleavage processes or the N-glycosidic bond breaking process. The only possible pathway that dominates the LEE-induced DNA single strands in the presence of the polarizable surroundings (such as in an aqueous solution) is the C_3′–O_3′ σ bond cleavage (the relatively low activation energy barrier, ∼13.4 kcal/mol, has been predicted through a polarizable continuum model investigation). The qualitative agreement between the ratio for the bond breaks of C_5′–O_5′, C_3′–O_3′ and N-glycosidic bonds observed in the experiment of oligonucleotide tetramer CGAT and the theoretical sequence of the bond breaking reaction pathways have been found. This consistency between the theoretical predictions and the experimental observations provides strong supportive evidences for the base-centered radical anion mechanism of the LEE-induced single-strand bond breaking around the pyrimidine sites of the DNA single strands.     

Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry

We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP-PC-Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5-position of 2′-deoxyribouridine 5′-triphosphate via a photocleavable 2-nitrobenzyl linker. We have demonstrated that dUTP-PC-Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP-PC-Biotin can be efficiently isolated by a streptavidin-coated surface and recovered by near-UV light irradiation at room temperature in mild condition for further analysis without using any chemicals or heat. Single and multiple primer extension reactions were performed using the dUTP-PC-Biotin to generate DNA products for MALDI-TOF MS analysis. Such nucleotide analogs that carry a biotin and a photocleavable linker will allow the isolation and purification of DNA products under mild conditions for MS-based genetic analysis by DNA sequencing or multiplex single nucleotide polymorphism (SNP) detection. Furthermore, these nucleotide analogs should also be useful in isolating DNA–protein complexes under non-denaturing conditions.     

On the DNA cleavage mechanism of Type I restriction enzymes

Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5′- and 3′-overhangs of varying lengths. EcoAI preferentially generated 3′-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5′-overhangs of a length of ~6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.