Nonradioactive detection of nucleic acid by the universal probe system

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.     

Applications of universal probe on DNA hybridization

A convenient method for DNA hybridization termed "Universal probe" is described which is based on the principle of sandwich hybridization. This system consists of two probes: primary probe which is single-stranded DNA prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target; and labeled secondary probe which has an opposite strand of the primary probe without the complementary sequence. By use this universal probe human beta-globin gene was able to be detected on Southern blots of genomic DNA. A potential advantage of this method is that the single-stranded primary probe is prepared easily by the chimeric phage-plasmid vector system and tedious labeling is not needed each time.     

A two-step hybridization method for chemiluminescent detection of single copy genes.

We have developed a technique for the chemiluminescent detection of single copy genes that eliminates the high backgrounds and problems with probe labeling associated with existing methods. The procedure employs a primary hybridization of single-stranded probe DNA to immobilized target DNA, a secondary hybridization with a covalently cross-linked oligonucleotide-alkaline phosphatase conjugate, followed by incubation in the chemiluminescent substrate AMPPD and detection on x-ray film. The key to the success of this method is that the primary probe contains a region complementary to the target DNA as well as to the oligonucleotide sequence of the secondary probe-alkaline phosphatase conjugate. Here we report our results using the two-step hybridization procedure to detect single copy genes from genomic Southern blots.     

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.     

P-loop catalytically assisting the enzymatic cleavage of single-stranded DNA

We demonstrated that a P-loop, a looped complex formed inside duplex DNA by adding peptide nucleic acids (PNA), acts catalytically as a template for enzymatic cleavage of single-stranded probe oligodeoxynucleotides (ODN). A PD-loop complex formed from P-loop and probe ODN was digested efficiently by a restriction enzyme, and the truncated probe ODN was released. The P-loop nicked by the enzyme can form PD-loop again with another probe ODN, and then assisted the enzymatic cleavage of an excess of probe ODN. In addition, by using dumbbell-formed ODN as a probe ODN, the efficiency of the P-loop-assisted ODN cleavage was enhanced considerably as compared with that of linear ODN. Thus, the method utilizing P-loop will make it possible to amplify the sequence information of duplex DNA via a catalytic cleavage of probe ODNs.     

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.     

Detection of DNA strand breaks in single cells using flow cytometry

A preliminary method is reported of alkaline unwinding of DNA within single cells and quantitation of the single-stranded and double-stranded DNA with the fluorescent probe acridine orange. A suspension of alkali-treated cells is obtained and analysed by flow cytometry. An increase in the amount of single-stranded DNA is taken as an indication of strand breaks. An advantage of this method is that a large number of cells can be individually analysed for DNA strand breaks. A measurement of DNA content is also obtained, making it possible to discriminate between cells in various parts of the cell cycle.     

A comparison of substrates for quantifying the signal from a nonradiolabeled DNA probe.

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.     

Directed covalent immobilization of aminated DNA probes on aminated plates

A new protocol that enables the immobilization of DNA probes on aminated micro-titer plates activated with aldehyde-dextran via an amino group artificially introduced in the 3' end of the oligonucleotide probe is reported in this work. The method is based on the use of hetero-functional-dextran as a long and multifunctional spacer arm covalently attached to an aminated surface capable of immobilizing DNA oligonucleotides. The immobilization occurred only via the amino introduced in the 3' end of the probe, with no implication of the DNA bases in the immobilization, ensuring that the full length of the probe is available for hybridization. These plates having immobilized oligonucleotide probes are able to hybridize complementary DNA target molecules. The tailor-made hetero-functional aldehyde-aspartic-dextran together with the chemical blocking of the remaining primary amino groups on the support using acetic anhydride avoid the nonspecific adsorption of DNA on the surface of the plates. Using these activated plates, (studying the effect of the probe concentration, temperature, and time of the plate activation on the achieved signal), thus, the covalent immobilization of the aminated DNA probe was optimized, and the sensitivity obtained was similar to that achieved using commercial biotin-streptavidin systems. The new DNA plates are stable under very drastic experimental conditions (90% formamide, at 100 degrees C for 30 min or in 100 mM NaOH).     

Preparation of single-stranded DNA from PCR products with streptavidin magnetic beads

The preparation of single-stranded DNA from double-stranded PCR products is an essential step in the identification of aptamers by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The most widely used method for producing single-stranded DNA is alkaline denaturation of biotinylated PCR products attached to streptavidin-coated magnetic beads. Recently, it has been suggested that this method may be unsuitable due to the release of interfering amounts of streptavidin and biotinylated DNA. In this article, the alkaline method is compared with a thermal method that is known to release significant amounts of streptavidin and biotinylated DNA. Results show that trace amounts of streptavidin and biotinylated DNA are released in the alkaline method, but this can be curtailed by preconditioning the beads in aqueous sodium hydroxide. The main product in the alkaline method is single-stranded DNA, which is produced in high yield.     

The effect of template secondary structure on vaccinia DNA polymerase.

Vaccinia virus DNA polymerase will utilize a substrate consisting of phi X174 DNA primed with a strand of a unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the phi X174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using Escherichia coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the phi X174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point of one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.     

Microwave irradiation-stimulated in situ hybridization procedure with biotinylated DNA probe.

A microwave-stimulated in situ hybridization technique using biotin-labeled DNA probe is described. Both hybridization reaction and the detection of the biotin label (with a alkaline phosphatase or immunofluorescence method) has been performed in the microwave oven. All procedures are completed within one hour. The described method was applied for identification of nucleic acid sequences of human immunodeficiency virus in human cell lines. The resolution and the intensity of the signal are as good as from a standard technique with overnight incubation of the probe. Because of the simplicity and speed of the technique, this procedure can be used in a number of other applications.     

Method enabling pyrosequencing on double-stranded DNA

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.     

Colorimetric detection of the tuberculosis complex using cycling probe technology and hybridization in microplates

Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA-DNA probe that is cut by RNase H when bound to its complementary target sequence. In this study, a hybridization assay was developed to detect biotinylated CPT products that result from the amplification of a Mycobacterium tuberculosis complex sequence. The chimeric probe was specifically designed to avoid the formation of secondary structures. The chosen capture probe was perfectly complementary to and was the same size as OL2, one of the two CPT products. The assay was based on the observation that a long sequence, such as the initial probe, was destabilized when bound to a small capture probe as a result of steric hindrance. The capture probe preferentially bound OL2 rather than the long initial probe. We added a prehybridization step with a helper DNA to enhance this discrimination between the two sequences. Colorimetric detection was performed using a peroxidase-streptavidin conjugate. After optimization, the non-isotopic hybridization assay allowed the detection of around 10 amol of target DNA. Besides being faster and easier to perform, this detection method was compared to electrophoresis separation and gave similar results.     

Characterization of alkaline phosphatase labeled UidA(Gus) probe and its application in testing of transgenic <Emphasis Type="Italic">tritordeum</Emphasis>

Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA–enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.     

Generation of a catalytic sequence-specific hybrid DNase

Hybrid nucleases consisting of an oligonucleotide fused to a unique site on the relatively nonspecific phosphodiesterase staphylococcal nuclease have been shown to sequence specifically cleave DNA. We have introduced mutations into the binding pocket of the nuclease which lower the kcat/Km of the enzyme. Hybrid nucleases generated from these mutants sequence selectively hydrolyze single-stranded DNA in a catalytic fashion, and under a much wider range of conditions than was previously possible. One such hybrid nuclease (Y113A, K116C) was able to site selectively cleave single-stranded M13mp7 DNA (7214 nt), primarily at one phosphodiester bond. Another hybrid nuclease (Y113A, L37A, K116C) catalyzed the hydrolysis of a 78-nt DNA substrate with a kcat of 1.2 min-1 and a Km of 120 nM. The effects of variations in the length and sequence of the oligonucleotide binding region were examined, as were changes in the length of the tether between the oligonucleotide and the enzyme. Cleavage specificity was also assayed as a function of substrate DNA primary and secondary structure and added poly(dA).     

Investigation into the effect that probe immobilisation method type has on the analytical signal of an EIS DNA biosensor

The analytical performance of an enhanced surface area electrolyte insulator semiconductor (EIS) device was investigated for DNA sensor development. The work endeavored to advance EIS performance by monitoring the effect of DNA probe layers have on the impedimetric signal during target hybridisation detection. Two universally employed covalent chemistries, direct and spacer-mediated attachment of amino modified probe molecules to amino-functionalised surfaces were investigated. Relative areal densities of immobilised probe were measured on planar and enhanced surface area substrates using epi-fluorescence microscopy. The reproducibility of the each immobilisation method was seen to have a direct effect on the reproducibility of the impedimetric signal. The sensitivity and selectivity was seen to be dependent on the type of immobilisation method. Real time, impedimetric detection of target DNA hybridisation concentrations as low as 25 and 1 nM were possible. The impact that probe concentration had on the impedimetric signal for selective and non-selective interactions was also investigated.     

Substrate specificity and mode of action of the zinc-metallo nuclease from Physarum polycephalum

The alkaline zinc-metallo nuclease of Physarum polycephalum is an endonuclease with a high specificity for single-stranded nucleic acids. Single-stranded DNA was cleaved at least 6,000 times faster than double-stranded DNA under identical conditions. In the supercoil-induced single-stranded region of Form I PM2 DNA only a single nick was made. The nuclease showed nucleotide specificity. Poly(A), poly(I), and poly(dT) were preferentially hydrolyzed. Product analysis showed that it acted by an endonucleolytic mechanism: long polynucleotides were fragmented via intermediate length products to oligo- and mono-nucleotides with the phosphate group at the 5'-terminal position. Extensive similarities exist with the single-strand-specific nuclease S1 from Aspergillus. The zinc-metallo endonuclease from Physarum could be used as a similar probe for single-stranded nucleic acids at neutral or alkaline pH conditions.