Evolutionary relationships of the Chinese hamster X chromosome and autosomes: a comparison using solution hybridization techniques

The evolutionary relationships of Chinese hamster X chromosome and autosome DNA sequences were compared by solution hybridization techniques. Chinese hamster X chromosome tracer was prepared by radiolabeling DNA from chromosomes isolated by fluorescence-activated sorting. Radiolabeled Chinese hamster total genomic DNA, approximately 90% of which is of autosome origin, was used as autosome tracer. Each tracer was mixed with excess driver DNA of Chinese hamster, Syrian hamster, rat, rabbit, cat, cow, or human origin. Reaction mixtures were melted and allowed to reassociate to an equivalent CoT of 12000, under conditions which permitted 35% mismatch in DNA duplexes. Both the extent of duplex formation (the normalized percentage hybridization or NPH) and the average thermal stability of the duplexes formed (melting temperature or Tm) were measured; these values were used to compare the evolutionary relatedness of tracer and driver DNAs. The pattern of evolutionary relatedness revealed by comparing either the Tm or NPH values obtained with different drivers was the same for X chromosome and autosome DNA and was consistent with the phylogeny of the species examined. Although NPH and Tm values for X chromosome and autosome tracers differed, differences fell within the range of experimental error. The results of these studies provide no evidence for differential conservation of Chinese hamster X chromosome sequences, suggesting that the constraints on the mammalian X chromosome which act to maintain its gene linkage group intact do not markedly reduce the extent to which its sequences diverge during evolution.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

A sensitive nonisotopic hybridization assay for HIV-1 DNA

We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

DNA hybridization detection on electrical microarrays using coulostatic pulse technique

We demonstrated a novel application of transient coulostatic pulse technique for the detection of label free DNA hybridization on nm-sized gold interdigitated ultramicroelectrode arrays (Au-IDA) made in silicon technology. The array consists of eight different positions with an Au-IDA pair at each position arranged on the Si-based Biochip. Immobilization of capture probes onto the Au-IDA was accomplished by self-assembling of thiol-modified oligonucleotides. Target hybridization was indicated by a change in the magnitude of the time dependant potential relaxation curve in presence of electroactive Fe(CN)(6)(3-) in the phosphate buffer solution. While complementary DNA hybridization showed 50% increase in the relaxation potential, the non-complementary DNA showed a negligible change. A constant behaviour was noted for all positions. The dsDNA specific intercalating molecule, methylene blue, was found to be enhancing the discrimination effect. The changes in the relaxation potential curves were further corroborated following the ELISA like experiments using ExtraAvidine alkaline phosphatase labelling and redox recycling of para-aminophenol phosphate at IDAs. The coulostatic pulse technique was shown to be useful for identifying DNA sequences from brain tumour gene CK20, human herpes simplex virus, cytomegalovirus, Epstein-Barr virus and M13 phage. Compared to the hybridization of short chain ONTs (27 mers), the hybridization of long chain M13 phage DNA showed three times higher increase in the relaxation curves. The method is fast enough to monitor hybridization interactions in milli or microsecond time scales and is well suitable for miniaturization and integration compared to the common impedance techniques for developing capacitative DNA sensors.     

A comparison of solution and membrane-bound DNA × DNA hybridization,as used to infer phylogeny

A new method of membrane-bound DNA × DNA hybridization was devised to accommodate the study of small quantities of DNA obtained from museum specimens for phylogeny reconstruction. Membranebound, single-stranded target genomic DNAs were competitively hybridized with a total genomic DNA probe to form hybrid duplexes required for the DNA dissociation experiments. We compared the thermal elution profiles derived from dissociating duplexes made with probes of whole genomic, single-copy, and repetitive DNA, as well as solution DNA × DNA hybridization using sc tracer. Quantitatively, pairwise indices of genetic distance derived from duplexes made with genomic probes depended entirely on hybridization of repetitive sequences, but a parallel set of experiments using repetitive and sc probes produced qualitatively similar results. The indices of genetic distance generated by the membrane-bound hybrids form an internally consistent, resolved tree which is in agreement with the solution DNA × DNA hybridization trials and traditional views of the phylogeny of the taxa under study.Correspondence to: P. Houde     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes

Summary A technique of in situ hybridization on metaphase chromosomes with biotinylated DNA probes is described. This technique was used to localize unique DNA sequences on chromosomes and allowed a localization of two probes 1.8 and 1.3 kb long. The hybridization signal appears like two, twin, spots on the two sister chromatids, allowing a clear distinction from the background. Moreover a chromosomal localization is possible by counting a relatively small number of mitoses compared with the technique using ³H-labeled DNA probes.     

Molecular hybridization of ribonucleic acid with a large excess of deoxyribonucleic acid

When RNA is annealed in solution with a sufficiently large excess of DNA, the kinetics of DNA-RNA hybridization are relatively simple. Methods are described for following the course of both DNA renaturation and DNA-RNA hybridization in this system. To explore the characteristics of the reaction a series of model systems was used. Each one utilized DNA (sheared to constant size) from a bacterium or bacteriophage and homologous cRNA, i.e. RNA synthesized in vitro on a template of the same DNA. Temperature optima were determined for the hybridization of Escherichia coli nucleic acids in 2xSSC and 3xSSC-50% formamide buffers, and of Proteus mirabilis nucleic acids in 2xSSC buffer. Rate-constants for DNA-RNA hybridization were measured by two methods. These gave somewhat different results, but in all cases the rate-constant of DNA-RNA hybridization was clearly less than that of DNA renaturation. Thus hybridization is a slower reaction than DNA renaturation. Nevertheless, in some cases, with a high concentration of DNA and a long annealing time, 90-95% of the added RNA became resistant to ribonuclease. Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA. Similarly, it is possible to estimate the reiteration frequency of multiple DNA sequences (such as ribosomal DNA) from the hybridization of the total DNA with RNA complementary to the multiple sequences. The effect on the system of various DNA/RNA ratios from 100 to 1 is described.     

Quantitative and qualitative analysis of amplified DNA sequences by a competitive hybridization assay.

The presence of allelic sequence variations in DNA fragments can be easily detected by measuring the extent of DNA strand exchange between test double-stranded PCR products (target) and labeled standard double-stranded PCR products (probe). Under selected hybridization conditions, sequences identical to the probe decreased the formation of double-labeled hybrid, whereas differing sequences were not efficient enough to compete with the regeneration of the probe. A single base substitution in the target DNA increased the percentage of remaining double-labeled probe. A general procedure involving denaturation and hybridization in solution under different temperature conditions or using different probes enabled sequence identification. The degree of regeneration of double-labeled probe was determined using a bioluminescent assay. We evaluated the specificity of this method with two probes (108 and 131 bp) and several targets with different base substitutions.     

Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization

We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.     

Analysis of whole-arm translocations in malignant blood cells by nonisotopic in situ hybridization

Whole-arm translocations in five leukemic patients were studied using nonisotopic in situ hybridization with alpha satellite DNA and simple satellite chromosome-specific DNA probes to detect the target sequences. The results show that this technique provides additional information on the involvement of these DNA sequences in whole-arm translocations.     

Carbon-nanotube-modified electrodes for amplified enzyme-based electrical detection of DNA hybridization

Carbon-nanotube-modified glassy carbon (CNT/GC) transducers have been developed for enhancing the sensitivity and stability of enzyme-based electrochemical bioassays of DNA hybridization. The amplified signal reflects the interfacial accumulation of phenolic products of the alkaline-phosphatase tracer onto the CNT layer. In particular, chronopotentiometric measurements (following short accumulation periods) offer a substantial enhancement of the response of enzymatically liberated alpha-naphthol. The CNT modifier also leads to a dramatic improvement in the stability of the amperometric response of alpha-naphthol. These advantages of CNT/GC electrodes are illustrated from comparison to unmodified glassy carbon electrodes. Factors influencing the adsorptive accumulation of alpha-naphthol, and the overall performance of the new DNA assay, are assessed and optimized. The attractive performance characteristics of the new multi-amplification electrochemical detection of DNA hybridization are reported in connection to the detection of nucleic acid sequences related to the breast cancer BRCA1 gene.     

Bi-color detection of two target DNAs by non-radioactive in situ hybridization

Summary A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.This investigation was supported in part by FUNGO, Foundation of Medical Scientific Research in The Netherlands (grant nr 13-54-21)     

A colorimetric method for DNA hybridization.

A general method has been developed which allows crosslinks to be produced between proteins and single-stranded DNA. Such single-stranded DNA protein complexes have been tested for blot hybridization using two colorimetrically detectable enzymes, namely peroxidase and alkaline phosphatase, as the protein moiety of the probe. After hybridization and incubation with a substrate solution sequences complementary to the probe can be visualized directly without the need of tedious cytochemical sandwich methods. This procedure will detect target sequences, a few kilobases long, in the 1- to 5-pg range.     

Study of DNA hybridization on polypyrrole grafted with oligonucleotides by photocurrent spectroscopy

Recognition of DNA sequences by biochemical sensor is generally performed by analysis after completion of hybridization. Using a technique able to directly translate the biological event into an electrical signal allows the in situ monitoring of the hybridization kinetics. In this aim, the photoelectrochemical behavior of one electroactive polymeric sensor based on a copolymer of polypyrrole and polypyrrole-oligonucleotide has been investigated in aqueous solution. This sensor has been studied as such (i) and in two other situations: (ii) when the copolymer is in presence of non-complementary oligonucleotides; and (iii) when the copolymer is in presence of complementary oligonucleotides. From the photocurrent spectra obtained at -0.6 V/SCE versus incident energy the allowed direct and indirect transitions for each polymer have been evidenced. The photocurrent evolution during hybridization and adsorption processes has been recorded in real time and the hybridization kinetics has revealed to be comparable with mass variations obtained by quartz crystal microbalance under the same experimental conditions.     

Use of pteridine nucleoside analogs as hybridization probes

The pteridine nucleoside analog 3-methyl isoxanthopterin (3-MI) is highly fluorescent, with a quantum yield of 0.88, and it can be synthesized as a phosphoramidite and incorporated into oligonucleotides through a deoxyribose linkage. Within an oligonucleotide, 3-MI is intimately associated with native bases and its fluorescence is variably quenched in a sequence-dependent manner. Bend ing, annealing, binding, digestion or cleavage of fluorophore-containing oligonucleotides can be detected by monitoring changes in fluorescence properties. We developed a single step method for detecting annealing of complementary DNA sequences using 3-MI-containing oligonucleotides as hybridization probes. One of the complementary strands contains the fluorophore as an insertion and when annealing occurs, the fluorophore bulges out from the double strand, resulting in increased fluorescence intensity. We have examined the sequence dependency, optimal strand length and impact of multiple fluorophores per strand in terms of brightness and impact on the annealing process. We describe the application of this technique to the detection of positive PCR products using an HIV-1 detection system. This sequence-dependent hybridization technique can result in fluorescence intensity increases of up to 27-fold. Fluorescence intensity increases are only seen upon specific binding to bulge-generating complements, removing issues of high background from non-specific binding.