Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.     

DNA sequencing by hybridization with an oligonucleotide matrix (SHOM). The theory of DNA elution after hybridization]

The theoretical treatment of the process of washing out of a single stranded DNA fragment after hybridization with short oligonucleotides immobilized within the polyacrylamide gel layer is presented. The theory describes satisfactorily the main body of experimental findings, obtained earlier in connection with the elaboration of a new DNA sequencing method based on hybridization with the matrix of immobilized oligonucleotides [K.R. Khrapko et al.@J. DNA Sequencing Mapp. 1991. V. 1. P. 373-388]. In particular the theory explains and describes well quantitatively the observed dependence of the "washing off temperature" Tw on the concentration of the immobilized oligonucleotides. The Tw dependence on inherent physico-chemical parameters such as the enthalpy and the entropy of duplex formation and on those selected by the experimenter (washing time duration, the gel thickness etc.) is also considered. A simple approximate expression for the calculation of "washing off temperature" is given. Some inconsistencies between calculated and observed washing curves are discussed.     

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.     

Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.     

Tag/hybridization-based sensitive detection of polymerase chain reaction products

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.     

DNA biosensors based on self-assembled carbon nanotubes

DNA biosensors based on self-assembled multi-walled carbon nanotubes (MWNTs) were described in this paper, in which the probe DNA oligonucleotides were immobilized by forming covalent amide bonds between carboxyl groups at the nanotubes and amino groups at the ends of the DNA oligonucleotides. Hybridization between the probe and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of the indicator of methylene blue. Our results demonstrate that the DNA biosensors based on self-assembled MWNTs had a higher hybridization efficiency compared to those based on random MWNTs. In addition, the developed DNA biosensors also had a high selectivity of hybridization detection.     

A method for DNA sequencing by hybridization with oligonucleotide matrix.

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.     

Attachment of oligonucleotide probes to poly carbodiimide-coated glass for microarray applications

Oligonucleotide-based DNA microarrays are becoming increasingly useful tools for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Here, we present a method that permits the manufacture of microarrays from non-modified oligonucleotides on a poly carbodiimide-coated glass surface by UV-irradiation. The use of UV-irradiation facilitates an increase in the level of signal intensity, but it does not affect signal discrimination by the oligonucleotides immobilized on the surface. The signal intensity obtained for an array fabricated using non-modified oligonucleotides with UV-irradiation is ~7-fold greater than that without UV-irradiation. The detection of SNPs was tested to ascertain whether this technique could discriminate specific hybridization signals without causing significant UV-irradiation-induced damage to the immobilized oligonucleotides. We found that this immobilization method provides greater hybridization signals and a better match/mismatch ratio of SNPs than do the established aminosilane techniques. Application of this technology to manufacturing DNA microarrays for sequence analysis is discussed.     

Enhancement of a hybridization analysis efficiency by the controlled DNA fragmentation

The influence of a DNA amplicon fragmentation on the efficiency of detecting a specific sequence by heterophase hybridization analysis was investigated. The nucleotide sequence was detected colorimetrically after biotinylation of an oligonucleotide probe immobilized on a solid carrier via limited elongation in complex with the sample DNA with the use of Taq polymerase. Two simple and reproducible techniques of amplicon fragmentation were proposed. The techniques are based on the introduction of apurinic/apyrimidinic sites in DNA and their subsequent thermal degradation. DNA was depurinated by a mild acidic treatment. Apyrimidinic sites were generated by treating a DNA fragment containing dUMP in place of some dTMP in various proportions with uracil-DNA glycosylase (UDG). The DNA sample treated by either method proved to be suitable for hybridization analysis with Taq polymerase without additional purification. The efficiency of hybridization analysis was higher with the fragmented than with the native DNA amplicon. DNA fragmentation makes it possible to use bridged oligonucleotides, having a lower hybridization ability, as highly selective hybridization probes.     

DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

A Fluorescent Dye with Low Specificity to DNA Nucleotide Sequences: Quantitative Assessment of Oligonucleotides Immobilized in Microchip Gel Pads

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)₈, d(T)₈, d(C)₈, and d(G)₈ at high temperatures (50°C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.     

Hybridization methods for DNA sequencing.

I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.     

DNA Sequencing by Hybridization to Oligonucleotide Matrix. Calculation of Continuous Stacking Hybridization Efficiency

Abstract

In this paper we consider the efficiency of additional rounds of “continuous stacking” hybridization in DNA sequence reconstruction by hybridization with oligonucleotide matrix (SHOM). After the initial hybridization of target DNA with the matrix of oligonucleotides of fixed length L some additional hybridizations should be carried out in the presence of fluorescently labeled oligonucleotides of another length l. These additional oligonucleotides can hybridize in tandem with matrix tuples (continuous stacking hybridization) thus forming an extended duplex with the target DNA strand. The additional data obtained allows resolutions of branching points arising in the reconstruction procedure. Multiple rounds of continuous stacking hybridization considerably increase the efficiency of the sequencing method, eventually approaching the power of (L+l)-matrix. We develop here an algorithm that allows us to minimize the number of additional hybridization steps, by assembling sets of l-tuples to be added together in each round of continuous stacking hybridization. For SHOM using a matrix of octanucleotides, continuous stacking hybridization with pen- tanucleotides increases the length of unambiguously sequenced DNA from 200 to several thousands of base pairs.     

Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Summary A fast, reproducible and non-hazardous technique for non-isotopic DNA fingerprinting is presented. The method is based on digoxigenated oligonucleotides, which are specific for simple repetitive DNA sequences. The use of digoxigenin/ anti-digoxigenin detection avoids many drawbacks inherent in e.g. the biotin/streptavidin system which often causes a poor signal-to-background ratio. Synthesis and purification of digoxigenated oligonucleotides and their use in filter hybridization are described in detail. Hybridization patterns obtained with four different radioactively labeled oligonucleotides have been compared with those of the respective digoxigenated probes. When slightly less stringent hybridization conditions are applied for digoxigenated oligonucleotides than for those labeled with ³²P, the signal intensities are satisfying but additional minor bands occur as a result of the reduced strigency. With one explainable exception, these bands increase the information content of the fingerprint. In addition, hybridization of the digoxigenated (CAC)₅ probe has been performed in situ with human metaphase chromosomes. The hybridization patterns in many mitoses resemble R-bands.     

Oligonucleotide-mediated, PCR-independent cloning by homologous recombination

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT).     

Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides.

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.     

A rapid method of gene detection using DNA bound to Sephacryl

A rapid method of gene detection has been developed utilising DNA fragments immobilized on resins and a sandwich hybridization assay. This method permits the detection of restriction fragment length polymorphisms (RFLPs) without the need to immobilize sample DNA. The method is based on the use of two non-overlapping DNA restriction fragments, one of which is attached to a resin (fragment A) and the other 32P-labelled (fragment B). Fragments A and B will not hybridize to each other unless there is a DNA or RNA fragment capable of hybridizing to both A and B present in the same reaction. Hybridization in this instance will result in the resin being radioactively labelled. The RFLP associated with the mutation causing sickle-cell anaemia was used as a model to develop the method. The resin Sephacryl S-500 appeared most suited to our method for two reasons: (i) DNA immobilization experiments using two coupling procedures and four resins indicated that Sephacryl S-500 bound the most DNA with very little non-covalent coupling. (ii) Hybridization experiments with DNA bound to a number of resins showed that DNA bound to Sephacryl S-500 hybridized most efficiently with a low level of nonspecific hybridization. Using optimum hybridization conditions 5 X 10(-18) mol of beta-globin DNA could be detected. The method has been used to distinguish between DNA from sickle, heterozygote and normal patients.     

Shielding effect of monovalent and divalent cations on solid-phase DNA hybridization: surface plasmon resonance biosensor study

Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg²⁺ in buffer led to significantly higher hybridization than even 1 M Na⁺). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.     

Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.