Detection and classification of Trypanosoma cruzi by DNA hybridization with nonradioactive probes.

Total or kinetoplast DNA (kDNA) from 72 isolates and clones of Trypanosoma cruzi as well as from nine related trypanosomatids were analyzed by dot hybridization using nonradioactive kDNA or cloned minicircle fragments as probes. Biotinylated-kDNA probes generated by nick-translation proved reliable for distinguishing Zymodeme 1 and Zymodeme 2bol of T. cruzi parasites. In contrast, digoxigenin-labeled kDNA obtained by random-priming did not distinguish among T. cruzi isolates but did distinguish among New World leishmanias. Cloned minicircle fragments labeled with digoxigenin gave the same results as digoxigenin-labeled kDNA, except for a 10-fold decrease in sensitivity. Digoxigenin-labeled DNA probes proved useful in unambiguously detecting T. cruzi from different geographic regions of America. However, T. rangeli and T. cruzi marinkellei were not distinguished by these probes.     

Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes.

A dot-blot hybridization immunoenzymatic assay with a chemiluminescent endpoint was developed for the rapid and sensitive detection of viral and plasmid DNAs. Digoxigenin-labeled probes were used to detect cytomegalovirus, parvovirus B19, and plasmid pBR328 DNAs. Hybridized probes were immunoenzymatically visualized by anti-digoxigenin Fab fragments labeled with alkaline phosphatase, and adamantyl 1,2-dioxetane phenyl phosphate was used as chemiluminescent substrate. Results were recorded by instant photographic films. The chemiluminescent hybridization assay was performed in about 8 hr and was able to detect as little as 50-10 fg of homologous target DNA.     

Use of non-radioactive digoxigenin-labeled DNA probes for RFLP analysis in rice

A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.     

A new method to perform nonradioactive tissue printing using digoxigenin-labeled riboprobes

We describe a modified protocol to perform a tissue print northern using digoxigenin-labeled riboprobes. The result of the hybridization is directly visualized on the membrane by an alkaline phosphatase detection system.     

Rapid typing of HLA-DQB1 DNA polymorphism using nonradioactive oligonucleotide probes and amplified DNA

The allelic sequence diversity at theHLA-DQBI locus has been analyzed by polymerase chain reaction (PCR) amplification and sequencing. Fifteen amino acid sequence-defined alleles (one previously unreported) and several silent nucleotide polymorphisms which subdivide these alleles have been identified. Here, we describe the specific amplification of theDQB1 second exon by several different PCR primer pairs and a simple and rapid typing procedure using a panel of 16 horseradish peroxidase (HRP)-labeled oligonucleotide probes capable of distinguishing theseDQBI alleles.     

Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes

A simple and rapid method for characterizing the polymorphism at the HLA-DPB1 locus has been developed. The procedure involves the selective amplification of the polymorphic second exon of the DPB1 locus by the polymerase chain reaction (PCR), followed by hybridization of the amplified DNA with 15 nonisotopic sequence-specific oligonucleotide probes. There are no sequences within the second exon of the DPB1 locus that uniquely define an allele; rather, each allele appears to arise from the shuffling of a limited number of polymorphic nucleotide sequences in six regions of variability. Consequently, individual alleles are identified by the pattern of hybridization of the 15 probes. Two formats for typing are described. In Format I (the dot-blot), the amplified DNA is ultraviolet (UV) cross-linked to a nylon membrane and hybridized with the oligonucleotide probes which are covalently labeled with horseradish peroxidase (HRP). In Format II (the reverse dot-blot), the oligonucleotides, which have poly-T tails, are bound to the membrane and the immobilized array of probes is hybridized to the PCR product which has incorporated biotinylated primers during the amplification process. In both formats, hybridization is detected by a simple colorimetric reaction. The application of this technology to the fields of tissue typing and individual identity is discussed. Offprint requests to: A. B. Begovich.     

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.     

Homologies between X and Y chromosomes detected by DNA probes: localisation and evolution.

We have isolated and characterized DNA probes that detect homologies between the X and Y chromosomes. Clone St25 is derived from the q13-q22 region of the X chromosome and recognizes a 98% homologous sequence on the Y chromosome. Y specific fragments were present in DNAs from 5 Yq-individuals and from 4 out of 7 XX males analysed. An X linked TaqI RFLP is detected with the St25 probe (33% heterozygosity) which should allow one to establish a linkage map including other polymorphic X-Y homologous sequences in this region and to compare it to a Y chromosome deletion map. Probe DXS31 located in Xp223-pter detects a 80% homologous sequence in the Y chromosome. The latter can be assigned to Yq11-qter outside the region which contains the Y specific satellite sequences. ACT1 and ACT2, the actin sequences present on the X and Y chromosomes respectively, have been cloned. No homology was detected between the X and Y derived fragments outside from the actin sequence. ACT2 and the Y specific sequence corresponding to DXS31 segregate together in a panel of Y chromosomes aberrations, and might be useful markers for the region important for spermatogenesis in Yq. Various primate species were analysed for the presence of sequences homologous to the three probes. Sequences detected by St25 and DXS31 are found only on the X chromosome in cercopithecoidae. The sequences which flank ACT2 detect in the same species autosomal fragments but no male specific fragments. It is suggested that the Y chromosome acquired genetic material from the X chromosome and from autosomes at various times during primate evolution.     

Gene probes.

Gene probes are used in virtually all disciplines of the life sciences. Probes will probably have their greatest impact in health care when used as diagnostic tools to detect and identify micro-organisms responsible for infectious disease. This review will use development of gene probes for the clinical laboratory as a theme and review recent patents and publications which have nurtured probe development.     

Screening expression libraries with nonradioactive immunological probes

An immunological screening method employing protein A-peroxidase which does not require radiolabelled antibodies for detection of Escherichia coli colonies synthesizing foreign proteins in a cDNA expression library is described. The technique is sensitive, simpler and more rapid than the procedures that rely on radiolabelled antibodies and autoradiography.     

Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.     

Preparation of nonradioactive probes for in situ hybridization

In situ hybridization (ISH) enables the precise localization of RNA targets and provides an avenue to study the temporal and spatial patterns of expression of specific genes. ISH has evolved from being an esoteric technique to one that is routinely used by researchers in many areas of research. A major driving force has been the development of numerous nonisotopic labeling and signal detection methods. Historically, radioactive probes and autoradiography provided sensitivity that was unattainable with nonisotopic probes. But the long exposure times required for signal detection and the perceived dangers associated with radioactivity limit its use. Advances in nonisotopic detection systems have overcome many of the limitations associated with using radiolabeled probes. One of the most significant contributions from nonisotopic methods is the ability to discriminate between multiple nucleic acid sequences simultaneously.     

Chemiluminescent nucleic acid detection with digoxigenin-labeled probes: a model system with probes for angiotensin converting enzyme which detect less than one attomole of target DNA.

Digoxigenin-labeled nucleic acid probes for angiotensin-converting enzyme were prepared by the polymerase chain reaction. In a model system, these probes were used to optimize conditions for target detection by direct chemiluminescence after dot-blot, Southern, or Northern hybridizations. Digoxigenin-labeled probes detected 23 fg (1.6 X 10(-19) mol or 9 X 10(4) copies) of target consistently.     

Metallothionein mRNA in the testis and prostate of the rat detected by digoxigenin-labeled riboprobe

Metallothionein (MT), a cysteine-rich heavy metal-binding protein, has been considered to play a role in the homeostatic control and detoxification of heavy metals, such as zinc, copper, and cadmium. In the present study, we have utilized a digoxigenin-labeled riboprobe to localize MT mRNA only by bright-field optics in the testis and prostate of the rat. In the rat testis, MT mRNA was found predominantly in primary spermatocytes and also in secondary spermatocytes and spermatids, but not in the spermatogonia, Sertoli cells, and Leydig cells. On the other hand, MT protein was present in these spermatogenic cells as well as in spermatozoa and Sertoli cells. In the prostate, MT mRNA was found predominantly in the epithelium of the dorsolateral lobes, but not in the ventral lobe, which is in agreement with the observed localization of MT protein. The utilization of both in situ hybridization and immunohistochemical staining on the same tissue specimens show MT gene expression in specific cell types in the male genital organs.     

Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes.

We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).     

Osmotic stress-induced genetic rearrangements in Escherichia coli H10407 detected by randomly amplified polymorphic DNA analysis

Randomly amplified polymorphic DNA (RAPD) analysis is a DNA polymorphism assay commonly used for fingerprinting genomes. After optimizing the reaction conditions, samples of Escherichia coli H10407 DNA were assayed to determine the influence of osmotic and/or oligotrophic stress on variations in RAPD banding patterns. Genetic rearrangements or DNA topology variations could be detected as changes in agarose gel electrophoresis banding profiles. A new amplicon generated using DNA extracted from bacteria prestarved by an osmotic stress and resuscitated in rich medium was observed. Enrichment improved recovery of mutator cells and allowed them to be detected in samples, suggesting that DNA modifications, such as stress-induced alterations and supercoiling phenomena, should be taken into consideration before beginning RAPD analyses.     

Unculturable microbes detected by molecular sequences and probes

Nucleic acid probes have revolutionized rapid diagnostics of plant-, animal- and human-pathogenic viroids, viruses, bacteria and protozoa, and have complemented the spectrum of immunological and serological tests. Sequences of genes and rRNA, of proven potential for identification of taxa, are a valuable tool in that both the identity and the phylogenetic position of an isolate can be elucidated. On the other hand, this approach is still laborious and requires sophisticated equipment. Designation of probes does not require information about sequences, although such knowledge is extremely helpful in the formulation of highly specific oligonucleotide probes, e.g. in the case of rDNA probes, where target stretches containing variable sequences are rather short (about 10–30 nucleotides). The application of probe technologies in molecular environmental studies byin situ hybridization, screening of gene libraries and flow cytometry is in its infancy but a logical step towards reconfirmation of the identity of a species in its natural habitat.