Deletion mapping of the testis determining locus with DNA probes in 46,XX males and in 46,XY and 46,X,dic(Y) females.

Eleven Y-specific DNA probes hybridizing with DNA from one or more 46,XX males were isolated from a recombinant phage DNA library constructed from flow sorted human Y chromosomes. Two probes hybridized with DNA from nine out of eleven, i.e. greater than 80% of these 46,XX males. The relative frequency of hybridization of the probes in the 46,XX males and in a 46,X,dic(Y) female, together with in situ hybridization data, allowed mapping of the probes on Yp in relation to a putative testis determining locus. Several of those probes were also absent in a 46,XY female, further refining a model for ordering the probes on Yp. The DNA of one XX male hybridized both with probes from Yp and probes from proximal Yq (excluding the pericentral region). This suggests that complex translocations may occur into the DNA of 46,XX males that involve not only parts of Yp but also parts of Yq.     

Development and Application of Cloned DNA Probes for Clavibacter xyli subsp. xyli, the Causal Agent of Sugarcane Ratoon Stunting

Eco RI restriction fragments of genomic DNA from Clavibacter xyli subsp. xyli (CXX) were ligated with plasmid pUC18 and cloned in Escherichia coli JM109. The cloned DNA inserts from recombinant plasmids were Eco RI-excised and labeled with non-radioactive digoxigenin and used as probes. Ten specific DNA probes, RSD3, 15, 30, 31, 32, 35, 37, 41, 71, and 73 were selected for disease detection and pathogen differentiation. In the specificity tests, all of the 10 CXX DNA, probes differentiated Clavibacter xyli from other bacteria specifically. Seven out of the 10 CXX probes crossreacted with C. x. subsp. cynodontis (CXC) very weakly under moderate stringency wash conditions of hybridization. In the sensitivity tests, all of the 10 DNA probes detected the homologous DNA of CXX from 0.19 to 0.75 ng. To detect various cell numbers of CXX, the DNA probes detected 10⁴ to 10⁵ cells effectively. In Southern hybridizations, distinctly different band patterns were shown when the probes hybridized with DNA from CXX and CXC. Among these probes, RSD3, 15, 30, 31, 35, 37, and 71, efficiently detected CXX present in the sap collected from symptomless sugarcane.     

Detection and identification of Mycoplasma infections by DNA hybridization

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.     

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.     

生物素标记HBV RNA探针的制备及应用

本文首次采用ＳＰ６５特殊质粒与人类的乙型肝炎病毒ＤＮＡ重组,制备了Ｂｉｏ－ＨＢＶ ＲＮＡ探针,能特异地与ＨＢＶ ＤＮＡ杂交,将该探针与缺口转移方法标记的Ｂｉｏ－ＨＢＶ ＤＮＡ探针进行了比较,结果显示出Ｂｉｏ－ＨＢＶ ＲＮＡ探针比Ｂｉｏ－ＨＢＶ ＤＮＡ探针的敏感性提高１０倍,并分别应用两种探针同时检测７０例乙肝病人血清中ＨＢＶ ＤＮＡ,阳性率各为３１．４２％、２８．５７％（Ｐ＞０．２５）。对Ｂｉｏ－     

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations

Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3' hydroxyl and 5' phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.     

Concerted evolution of alpha satellite DNA: Evidence for species specificity and a general lack of sequence conservation among alphoid sequences of higher primates

We investigated relationships among alpha satellite DNA families in the human, gorilla, chimpanzee, and orangutan genomes by filter hybridization with cloned probes which correspond to chromosome-specific alpha satellite DNAs from at least 12 different human chromosomes. These include representatives of both the dimer-based and pentamer-based subfamilies, the two major subfamilies of human alpha satellite. In addition, we evaluated several high-copy dimer-based probes isolated from gorilla genomic DNA. Under low stringency conditions, all human probes tested hybridized extensively with gorilla and chimpanzee alpha satellite sequences. However, only pentameric and other non-dimeric human alphoid probes hybridized with orangutan alpha satellite sequences; probes belonging to the dimer subfamily did not cross-hybridize detectably with orangutan DNA. Moreover, under high stringency conditions, each of the human probes hybridized extensively only with human genomic DNA; none of the probes cross-hybridized effectively with other primate DNAs. Dimer-based gorilla alpha satellite probes hybridized with human and chimpanzee, but not orangutan, sequences under low stringency hybridization conditions, yet were specific for gorilla DNA under high stringency conditions. These results indicate that the alpha satellite DNA family has evolved in a concerted manner, such that considerable sequence divergence is now evident among the alphoid sequences of closely related primate species.     

Localization of the structural gene for botulinum neurotoxin type A using synthetic DNA probe for neurotoxin light chain

To obtain the information on the genetic control of toxin production in the botulism causative agents, the oligonucleotides were synthesized as the molecular probes by translation of the amino acid sequence of the botulinic type A neurotoxin. The optimal conditions for hybridization of botulinic DNA with the synthetic DNA probes were determined and the probes specificity was demonstrated. The DNA fragments homologous to the probes used were shown to belong to bacterial genome, but not to bacteriophage one.     

Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.     

Chromosomal localization of intergenomic RFLP loci in hexaploid wheat.

Hybridization of radiolabeled wheat DNA probes to genomic DNA digests of compensating nullisomic-tetrasomic lines and ditelosomic lines of hexaploid wheat (Triticum aestivum L. cv. Chinese Spring) can be used to identify intergenomic RFLPs. Sixty-three PstI/BamHI genomic DNA probes and eight cDNA probes were used to determine the chromosomal locations of 223 DNA fragments that define a minimum of 189 RFLP loci. Eighty-four percent of the genomic DNA clones hybridize to fragments located in homoeologous chromosomes and 16% hybridize to fragments located in one chromosome only or to fragments located in nonhomoeologous chromosomes. All of the cDNA probes hybridize to fragments located in homoeologous chromosomes.     

Preparation of synthetic tandem-repetitive probes for DNA fingerprinting

DNA fingerprints are generated using probes that hybridize to hypervariable minisatellites, also known as variable number tandem repeat loci. Cloned minisatellites have served as the predominant source of DNA fingerprinting probes. A short segment within the repeat units of minisatellites, called the "core" sequence, is highly conserved within a family of related minisatellites, thereby allowing a single-cloned minisatellite to cross-hybridize to 20 to 40 other minisatellites. In this article, we describe a method for the synthetic preparation of polymeric core sequence probes for DNA fingerprinting. Unlike "monomeric" oligonucleotide probes, the polymeric probes mimic the tandem-repetitive structure of minisatellites, and thus each probe molecule can potentially form many sites of hybridization with a target minisatellite. The synthetic probes are cloned into plasmid DNA to provide a perpetual source of probe material.     

Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.     

Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.     

The use of arbitrarily primed PCR (AP-PCR) to develop taxa specific DNA probes of known sequence

Abstract A rapid polymerase chain reaction (PCR) method to obtain taxa-specific DNA probes has been developed. The oligonucleotide probes derived from the sequences of species-specific (or other taxa) random amplified polymorphic DNA (RAPD) fragments. The methodology was applied to design probes for the halophilic archael species Haloferax mediterranei . With this technique, DNA probes of known sequence can be generated easily and without any previous knowledge about the properties of the microorganisms.     

DNA fingerprinting of eukaryote genomes by synthetic oligodeoxyribonucleotide probes

An extreme level of DNA sequence polymorphism, the basis of DNA fingerprinting, was first demonstrated using genome derived cloned probes. Subsequently, it was shown that DNA fingerprinting can also be carried out using short synthetic oligodeoxyribonucleotide probes specific for simple repetitive sequences. Further, in addition to radioactively labeled probes, non-radioactive oligonucleotides generate equally informative hybridization patterns. We discuss the development in the area of DNA fingerprinting and its future scope with respect to plant, animal and the human DNA.     

Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples.

Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples.     

Coupling into the base pair stack is necessary for DNA-mediated electrochemistry

The electrochemistry of DNA films modified with different redox probes linked to DNA through saturated and conjugated tethers was investigated. Experiments feature two redox probes bound to DNA on two surfaces: anthraquinone (AQ)-modified uridines incorporated into thiolated DNA on gold (Au) and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-modified uridines in pyrene-labeled DNA on highly oriented pyrolytic graphite (HOPG). The electrochemistry of these labels when incorporated into DNA has been examined in DNA films containing both well matched and mismatched DNA. DNA-mediated electrochemistry is found to be effective for the TEMPO probe linked with an acetylene linker but not for a saturated TEMPO connected through an ethylenediamine linker. For the AQ probe, DNA-mediated electrochemistry is found with an acetylene linker to uridine but not with an alkyl chain to the 5' terminus of the oligonucleotide. Large electrochemical signals and effective discrimination of intervening base mismatches are achieved for the probes connected through the acetylene linkages, while probes connected through saturated linkages exhibit small electrochemical signals associated only with direct surface to probe charge transfer and poor mismatch discrimination. Thus DNA electrochemistry with these probes is dramatically influenced by the chemical nature of their linkage to DNA. These results highlight the importance of effective coupling into the pi-stack for long-range DNA-mediated electrochemistry.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.     

Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.