A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

N-(Iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED): a new heterobifunctional reagent for the preparation of biochips

Design and synthesis of a new heterobifunctional reagent, N-(iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED), have been described for the preparation of oligonucleotide-based biochips. The performance of the featured reagent is probed by the immobilization of thiolated and thiophosphorylated oligonucleotides on modified glass microslides via two routes (routes A and B). The immobilization procedure was accelerated by performing a chemical reaction between thiolated oligomers and the iodoacetyl moiety of the reagent under microwaves (MW), where it is completed in just 10 min. The quality of the constructed oligonucleotide microarrays was tested by performing a hybridization assay with a complementary target and subsequently used for the detection of base mismatches. The immobilized probes were found to be thermally stable.     

N-(3-Trifluoroethanesulfonyloxypropyl)anthraquinone- 2-carboxamide: a new heterobifunctional reagent for immobilization of biomolecules on a variety of polymer surfaces

A new heterobifunctional reagent, N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) has been developed, useful for making bioconjugates and immobilization of biomolecules, viz., oligonucleotides, peptides, proteins, etc., on a variety of carbon-containing solid surfaces. Its trifluoroethanesulfonate ester group reacts with aminoalkyl or mercaptoalkyl functions present in biomolecules, and the anthraquinone structure reacts with a variety of carbon-containing polymers under ultraviolet irradiation (365 nm). The reagent has been used in two ways. First, the reagent, NTPAC, was first brought in contact with the above said supports and exposed to long wavelength ultraviolet light (365 nm), thereby generating active trifluoroethanesulfonate ester functions on the support, which subsequently react with appropriate mercaptoalkyl- or aminoalkyl-containing biomolecules to fix them on the supports. In another route, the proposed reagent was allowed to react first with proteins or 5'-aminoalkyl- or mercaptoalkyl-modified oligonucleotides to form the appropriate biomolecule-anthraquinone conjugate, which was then brought in contact with a variety of carbon-containing polymers, viz., modified controlled pore glass (CPG), modified glass microslides, cross-linked polystyrene, nylon, cross-linked polysaccharides, polypropylene (PP), polyethylene (PE), etc., and exposed to long wavelength ultraviolet light (365 nm), resulting in immobilization of the conjugates on the support. Both of the routes work satisfactorily and we could successfully immobilize a number of enzymes and modified oligonucleotides on a variety of supports.     

N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC): a heterobifunctional reagent for immobilization of biomolecules on glass surface

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide (TPICC) has been developed by a simple 'two step reaction' for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides and proteins on the glass surface. The isothiocyanate functionality at one end of the reagent, TPICC was found specific for the ligands having either aminoalkyl (RNH(2)) or mercaptoalkyl (R-SH) functionality. The synthesis of bioconjugates with the reagent was achieved through its isothiocyanate functionality at one end via the formation of stable thiourea linkage with aminoalkyl and dithiocarbamate linkage with mercaptoalkyl derivatives. The triethoxysilyl functionality of the reagent has been utilized for specific coupling with the virgin glass surface by a very simple methodology.     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

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.     

Microarray fabrication with covalent attachment of DNA using bubble jet technology

We have developed a method for fabricating DNA microarrays that uses a Bubble Jet ink jet device to eject 5'-terminal-thiolated oligonucleotides to a glass surface. The oligonucleotides are covalently attached to the glass surface by heterobifunctional crosslinkers that react with the amino group on the substrate and a thiol group on the oligonucleotide probe. Using this method, we fabricated DNA microarrays that carried 64 groups of 18-mer oligonucleotides encoding all possible three-base mutations in the mutational "hot spot" of the p53 tumor-suppressor gene. These were screened with a fluorescently labeled synthetic 18-mer oligonucleotide derived from the p53 gene, or segments of the p53 gene that had been PCR amplified from genomic DNA of two cell lines of human oral squamous cell carcinoma (SCC). This allowed us to discriminate between matched hybrids and 1 bp-mismatched hybrids.     

Immobilization of oligonucleotides on poly(ethylene glycol) brush-coated Si surfaces

High-density poly(ethylene glycol) (PEG) molecules are grafted onto Si surfaces in a brush-like configuration. We demonstrate that this surface is an excellent substrate for oligonucleotide immobilization. p-Maleimidophenyl isocyanate is used as a heterobifunctional cross-linker to tether thiol-modified oligonucleotides to terminal OH groups on the PEG brush. This approach gives excellent immobilization specificity and low background. The immobilized oligonucleotides show high sensitivity for the detection of complementary targets.     

A facile method for the construction of oligonucleotide microarrays

In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.     

Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides

A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self-quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluorophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies.     

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm² from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.     

Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5'-protected thiol function and a 3'-amino group

A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5′-ends and an amino group at their 3′-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3′-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2′-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3′-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5′-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3′-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligonucleotide; the 5′-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an Nα-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate.     

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected ω-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.     

Characterization of oligodeoxyribonucleotide synthesis on glass plates

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5′-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using ³²P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5′-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.     

A rapid method for the construction of oligonucleotide arrays

A simple method has been devised to construct oligonucleotide array on a variety of surfaces using commonly available reagents and chemistry with good efficiency and accuracy. The method involves the generation of hydroxyl functionalities on glass, polypropylene, polyethylene, and commonly used surfaces for construction of oligonucleotide arrays followed by their activation with trifluoroethanesulfonyl chloride (tresyl chloride). The activated surface in the subsequent reaction is used to covalently immobilize oligonucleotides in regioselective fashion to create an oligonucleotide array. The surface bound tresyl sulfonate esters allow the immobilization of oligonucleotides specifically via their 3'- or 5'-end having mercaptohexyl- or aminohexyl functionalities. The constructed oligonucleotide arrays were successfully used to analyze oligonucleotides by hybridization technique.     

In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication

In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 μm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling. High, reproducible yields were achieved for both 6mer and 21mer probes as assessed by hybridization to fluorescent oligonucleotides. Oligonucleotide surface density was comparable with that obtained on glass substrates. These results suggest PDMS as a stable and flexible alternative to glass as a suitable substrate in the fabrication and synthesis of DNA microarrays.     

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.     

Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5′ end adjacent to the single-strand–duplex junction. Deletion of the free 3′ end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3′ end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5′ end failed to block cleavage, although this substrate was refractory to cleavage by the 5′-3′ exonuclease activity of Taq DNA polymerase. For verification, the free 5′ end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5′ single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion X174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.     

Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.