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PubMed1986

Genetic relatedness among strains of the Mycobacterium tuberculosis complex. Analysis of restriction fragment heterogeneity using cloned DNA probes.

Chromosomal DNA isolated from 3 reference and 2 clinical isolates of Mycobacterium tuberculosis, 2 reference strains each of M. bovis and M. bovis BCG, and 1 reference strain of M. kansasii, was digested with 9 restriction endonucleases. The restriction fragments were analyzed by agarose gel electrophoresis. With each enzyme tested the patterns of the strains of M. tuberculosis, M. bovis, and M. bovis BCG were indistinguishable but clearly distinct from those of M. kansasii. A library of M. tuberculosis H37Rv DNA was prepared by cloning Bam HI digest fragments into lambda 1059. Eight randomly selected clones, having multiple Bam HI sites, were used to probe restriction digests of DNA from the strains of the tuberculosis complex. Two of the cloned segments hybridized with homologous fragments in 4 different enzyme digests of all strains. With 2 clones, hybridization differed among the strains; however, some homologous sequences were detected. With 4 clones, efficient hybridization occurred only with M. tuberculosis H37Rv DNA. These hybridization results indicate that some regions of the chromosome are highly conserved among members of the tuberculosis complex, whereas others are diverged. Selected DNA probes were useful in detecting differences among strains of the tuberculosis complex.

Cloning, Molecular↗

PubMed1994

Genotypic characterization of rickettsiae by DNA probes generated from Rickettsia prowazekii DNA.

Southern blot analysis of HindIII-cleaved rickettsial DNA was used for genotypic characterization of the typhus group (TG) species (R. prowazekii, R. typhi, R. canada) and a few species of the spotted fever group (SFG) rickettsiae (R. sibirica, R. conorii, R. akari). Four different DNA probes were employed. PBH11 and PBH13 probes were morphospecific HindIII fragments of R. prowazekii DNA. MW218 probe contained the gene for 51 K antigen and MW264 probe contained the citrate synthase gene of R. prowazekii. All the probes hybridized with the tested TG and SFG rickettsial DNAs, forming from 1 to 5 bands, but they did not with R. tsutsugamushi or C. burnetii DNAs. All the probes demonstrated specific hybridization patterns with TG species and R. akari. PBH11, PBH13 and MW264 probes clearly distinguished R. sibirica and R. conorii from the other tested rickettsiae, but not from each other. However, these two species differed slightly with MW218 probe. Several strains of each species were analyzed in this way and except for strains of R. conorii identical intraspecies patterns were obtained. These data lead us to consider the obtained hybridization patterns as criteria for genotypic identification.

Animals↗

PubMed1992

Efficient cDNA cloning method using solid phase DNA probe.

We are now developing a novel and efficient method using solid phase DNA probe to isolate a particular recombinant cDNA from single stranded cDNA library. Target clone coding metapyrocatechase (MPC) and cDNA library constructed from mRNA of U-937 (human lymphoma cell line) were converted to single stranded form by superinfection of helper phage (M13KO7). Probe DNA (25 mer) composed of a portion of the target cDNA was synthesized, attached to an HPLC gel and used as a solid phase DNA probe. Hybridization between probe DNA and target clone was performed in an Eppendorf tube within a few hours. Competent cell (JM109) was transformed with about one-twentieth of hybridized and eluted fraction by Hanahan's method. From the mixture of 1 ng of MPC vector and 5 micrograms of cDNA library, we obtained 50 colonies containing MPC gene out of 63 transformed colonies.

Catechol 2,3-Dioxygenase↗

PubMedDate not supplied

Detection of infectious laryngotracheitis virus in chickens using a non-radioactive DNA probe.

A DNA hybridization assay using a non-radioactive probe has been developed for the detection of infectious laryngotracheitis virus (ILTV) DNA. A 1.4-kilobase pair BamHI fragment of ILTV genomic DNA was cloned and then labeled by one of two methods; nick translation using 32P-dATP or non-radioactive labeling using a commercially available DNA labeling and detection kit. The non-radioactive DNA labeling method proved to be as sensitive as the radioactive method. Using the non-radioactive probe, ILTV DNA was readily detected in tracheal samples from acutely infected chickens and also from convalescent chickens at a time when viral antigen could no longer be detected by the enzyme-linked immunosorbent assay or the virus could no longer be reisolated. This technique provides a safe and effective means of identifying field outbreaks of ILTV and also may detect latent ILTV infections relatively quickly and inexpensively.

Animals↗

PubMed1992

Fluorescence in situ hybridization (FISH) of a whole-arm translocation involving chromosomes 18 and 20 with alpha-satellite DNA probes: detection of a centromeric DNA break?

Fluorescence in situ hybridization (FISH) with alpha-satellite DNA probes was used to study whole-arm chromosome translocation products in a family in which the propositus was shown to have a monosomy 18p/trisomy 20p imbalance. By this approach, we show that the chromosome 18 alpha-satellite DNA block is split into 2 smaller units, whereas the chromosome 20 breakpoint is not included within the alpha-satellite DNA region. We found no evidence to suggest that this split alpha-satellite DNA region has reduced or impaired the function of the centromere or that it contributed to the phenotype of the propositus. The FISH technique critically demonstrated the involvement of a whole-arm translocation in this case and provided accurate identification of breakpoints, which was not possible with standard banding techniques.

Abnormalities, Multiple↗

PubMed1990

Genetic mapping and characterization of sorghum and related crops by means of maize DNA probes.

Cloned DNA fragments from 14 characterized maize genes and 91 random fragments used for genetic mapping in maize were tested for their ability to hybridize and detect restriction fragment length polymorphisms in sorghum and other related crop species. Most DNA fragments tested hybridized strongly to DNA from sorghum, foxtail millet, Johnsongrass, and sugarcane. Hybridization to pearl millet DNA was generally weaker, and only a few probes hybridized to barley DNA under the conditions used. Patterns of hybridization of low-copy sequences to maize and sorghum DNA indicated that the two genomes are very similar. Most probes detected two loci in maize; these usually detected two loci in sorghum. Probes that detected one locus in maize generally detected a single locus in sorghum. However, maize repetitive DNA sequences present on some of the genomic clones did not hybridize to sorghum DNA. Most of the probes tested detected polymorphisms among a group of seven diverse sorghum lines tested; over one-third of the probes detected polymorphism in a single F2 population from two of these lines. Cosegregation analysis of 55 F2 individuals enabled several linkage groups to be constructed and compared with the linkage relationships of the same loci in maize. The linkage relationships of the polymorphic loci in the two species were usually conserved, but several rearrangements were detected.

Blotting, Southern↗

PubMed1991

[Paternity test with single locus DNA probes].

Seven kinds of DNA probes recognizing hypervariable DNA loci were applied to 28 cases of paternity test, involving two cases in which the putative fathers had died. The combinations of probe and restriction enzyme are as follows; MR24/1-HinfI, 3'Globin-PvuII, Ha-ras-PvuII, Mucin-PvuII, D2S44 (pYNH24)-MspI, D17S30 (pYNZ22)-MspI, D1S57 (pYNZ2)-RsaI. The reported number of the alleles are 37, 39, 5, 10, 33, 15, and 5, respectively. Those probes lie on different chromosomes except D1S57 on 1p and Mucin on 1q21. Exclusion probability (EP) and paternity index (PI) were calculated from the allele frequencies in Japanese population reported by Yokoi et al. Cumulative EP from 7 DNA probes was 0.999932, and cumulative PI ranged from 7.3 X 10(6) to 947. Also, cumulative EP from 17 kinds of conventional blood group markers (CBGM) was 0.9776, and cumulative PI ranged from 1290 to 0.11. Total EP from 7 DNA probes and CBGM was 0.999998478. Cumulative PI from 7 DNA probes were 5 to 2,000,000 times higher than that from CBGM. The single locus hypervariable DNA polymorphisms are considered to be informative for paternity test.

DNA Probes↗

PubMed1990

Identification of atypical strains of Streptococcus pneumoniae by a specific DNA probe.

A specific DNA probe containing a 0.65 Kb fragment coding for the aminoterminal region of the major pneumococcal autolysin (amidase) was constructed, deleting the region involved in the specific recognition of cell wall choline residues. The high specificity of this probe was demonstrated in tests with Streptococcus pneumoniae related species including Streptococcus oralis, which contains choline in its cell wall. The probe was used to characterize pneumococcal isolates showing atypical responses in conventional identification tests. The hybridization obtained with 27 atypical pneumococci and 11 of 17 isolates presumptively identified as viridans streptococci confirmed that the probe is suitable for diagnostic use.

DNA Probes↗

PubMed2000

Genetic polymorphism in Xanthomonas albilineans strains originating from 11 geographical locations, revealed by two DNA probes.

Two DNA fragments from Xanthomonas albilineans were used as probes to study the molecular diversity among strains of this pathogen. Two serologically distinct groups, serovars I and II, could be differentiated by hybridization to the probes. These probes, designated 830 and 838, were cloned after subtractive DNA hybridization of common sequences of Xanthomonas campestris pv. vasculorum from a serovar I strain of X. albilineans. They did not hybridize to the DNA of several other xanthomonads or to sugarcane DNA under the conditions of hybridization used. Faint bands were observed upon hybridization of probe 830 with one strain of X. campestris pv. phaseoli. The same banding patterns were obtained with a strain of X. albilineans from Burkina Faso and the serovar II strains of Mauritius. The serovar I strains from Mauritius and two other strains each from Reunion and South Africa had similar pattern.

Africa↗

PubMed1997

Detection of Babesia bovis using a DIG-labeled DNA probe.

A plasmid DNA containing the inserted Babesia bovis cDNA clone designated c51A was used to prepare a DNA probe for B. bovis. The purified 0.6 kb specific DNA fragment was labeled by DIG DNA labeling. After denaturation, the probe was hybridised with the blotted target DNA extracted from bovine red blood cells infected with B. bovis or other protozoa or bovine red blood cells plus extra white blood cells. It was found that the probe produced from purified 0.6 kb DNA fragment could detect sample of B. bovis DNA equivalent to 0.015 microliter of 10% whole infected blood. Compared to the same DNA fragment labeled with photobiotin, this new probe is more sensitive giving a darker hybridization signal, a lighter hybridization background and without any non-specific reactions. These results indicated that this DIG-labeled B. bovis C5lA probe could provide a sensitive and specific method to diagnose clinically suspected B. bovis infections and distinguish B. bovis DNA from other haemoprotozoan infections.

Affinity Labels↗

PubMed1989

Human identification and sex determination of dental pulp, bone marrow and blood stains with a recombinant DNA probe.

Recombinant DNA hybridizing specifically to a 300 nucleotide repeat DNA sequence (BLUR8) of human specificity and to human repeat DNA sequence (pHY10) on the Y chromosome was used for human identification and sex determination of degraded DNA samples of blood stains, dental pulp, and bone marrow. This radioactive technique enabled reliable and sensitive human and sex determination from blood stains that were more than 80 years old. Less than 1 piece of 0.5 cm length thread of blood stain was enough for both tests. DNA from relatively fresh dental pulp and bone marrow was clearly identified. The human identification test, which could recognize up to 0.3 ng DNA correctly, was 3 to 5 times more sensitive than the sex determination test.

Adolescent↗

PubMed1992

Quantitation of changes in mitochondrial DNA during aging and regeneration of rat liver using non-radioactive DNA probes.

By using DNA probes prepared from cloned cells which contain mitochondrial DNA (mtDNA) sequences in plasmids, the specific detection of mtDNA became possible in the presence of large excess of nuclear DNA by DNA-DNA hybridization. For this purpose, we prepared mtDNA probes labeled with non-radioactive substrate, which allowed a wider possibility of application. This method revealed that the contents of mtDNA in rat liver are strikingly decreased during aging. Furthermore, it was observed that although mtDNA content increased upon partial hepatectomy even in old rats, it decreased to the pre-operation level rather rapidly within 1 week after reaching peak in regenerated liver.

Aging↗

PubMed1988

Differentiation of Plasmodium falciparum clones by means of a repetitive DNA probe.

A recombinant DNA probe, pBRK1-14, was constructed by inserting a repetitive DNA fragment of 753 base pairs obtained from Plasmodium falciparum, isolate K1, into the EcoRI/PstI cloning sites of pBR322. This probe could discriminate between different parasite clones derived from a single isolate by hybridization with Southern genomic blots.

Animals↗

PubMed1994

Probing for answers. Use of DNA probes in infectious diseases.

Diagnostic molecular biology is at the forefront in the diagnosis of infectious diseases. Through the use of deoxyribonucleic acid (DNA) probes in conjunction with other molecular techniques such as polymerase chain reaction (PCR), physicians will be able to more rapidly and accurately diagnose causative agents in a variety of infectious diseases. This article will focus on the molecular basis of these techniques, how they may aid the physician in patient diagnosis, and the shortcomings of this new and exciting technology.

Communicable Diseases↗

PubMed2004

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.

DNA↗

PubMedDate not supplied

Biocontrol strain Pseudomonas sp. W34: specific detection and quantification in the rhizosphere of Cucumis sativus with a DNA probe and genotypic characterization by DNA fingerprinting.

A DNA probe specific for biocontrol strains of Pseudomonas was produced by screening randomly amplified polymorphic DNA (RAPD) PCR fragments. Specificity of the probe was assessed by dot blot and colony hybridization. It was used to specifically determine the population of these strains on roots of Cucumis sativus cv. Delikatess. Two polymorphic RAPD fragments of 750 bp, and 550 bp showed identical specificity. The biocontrol strain Pseudomonas sp. W34 was shown to be competitive in the rhizosphere of cucumber and to maintain a stable population for at least 10 days when inoculated on the seed. The phylogenetic relationships between the biocontrol and reference strains were analyzed at the strain level by means of RAPD and repetitive sequence-based PCR genomic fingerprinting (rep-PCR), and at higher taxonomic levels by means of amplified 16S ribosomal DNA restriction analysis ARDRA. It was shown that the antagonistic strains are closely related, forming a separate cluster from other non-antagonistic and reference Pseudomonas strains, their taxonomic placement remaining uncertain.

Cucumis sativus↗

PubMed1987

Comparison of genomic, plasmid, synthetic, and combined DNA probes for detecting Plasmodium falciparum DNA.

Total genomic Plasmodium falciparum DNA, the plasmid clone pRepHind, and a 21-base-long synthetic DNA probe (PFR1), the sequence of which was derived from pRepHind, were hybridized with DNA from various species of the phylum Apicomplexa. The genomic probe hybridized with P. reichenowi and P. falciparum DNA and significantly cross-hybridized with DNA of all the other Plasmodium species tested. The synthetic and plasmid probes hybridized to P. falciparum DNA and at reduced levels to P. reichenowi but did not hybridize to P. vivax, P. malariae, P. ovale, P. fragile, P. inui, P. knowlesi, Babesia bovis, B. microti, B. bigemina, Anopheles sp., Pan sp., Aotus sp., or human DNA. Southern blot analysis indicated that approximately 60 distinct restriction enzyme fragments from P. falciparum DNA were similarly detected by PFR1 and pRepHind. A method was developed by using a second brief hybridization with synthetic DNA to amplify signals from samples that were previously hybridized with plasmid-borne repetitive DNA. This amplification procedure was shown to allow the detection of 0.005% P. falciparum parasitemias from 10-microliter samples of blood from patients in Kenya.

Animals↗

PubMed1997

Comparisons of cost and accuracy of DNA probe test and culture for the detection of Neisseria gonorrhoeae in patients attending public sexually transmitted disease clinics in Los Angeles County.

BACKGROUND: Strict handling and transport requirements for the successful use of culture in the detection of Neisseria gonorrhoeae warrant investigation of accurate and cost-effective test alternatives such as the Gen-Probe PACE 2 DNA probe assay (Gen-Probe, Inc., San Diego, CA). STUDY DESIGN: The Gen-Probe PACE 2 DNA probe assay for N. gonorrhoeae was compared with conventional culture methods in the principal Los Angeles County (LAC) Department of Health Services (DHS) Public Health Laboratory and three of its branch laboratories. Urethral and endocervical samples were collected from 1,566 patients (921 males; 645 females) attending six LAC DHS sexually transmitted disease clinics. Cost analysis was performed comparing material and labor costs of the two test methods. RESULTS: The overall prevalence based on culture was 11.8% (15.7% for males; 6.4% for females). Nine samples were culture positive, Gen-Probe negative and four samples were culture negative, Gen-Probe positive and remained discordant after discrepant analysis. The sensitivity and specificity were 94.6% and 99.7%, respectively, for the PACE 2 assay compared with culture. The positive and negative predictive values were 97.8% and 99.3%, respectively. No statistically significant difference was found between the two tests. A cost analysis found an average cost of $3.11/test for culture and $3.85/test for PACE 2, given the approximate 12% disease prevalence in this population. CONCLUSIONS: Gen-Probe's PACE 2 assay may provide an acceptable, cost-effective alternative to culture, especially among high-risk males.

Adult↗