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At least 325 records · Page 18Linked to original sources
PubMed1998

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

Animals↗
PubMed1998

Detection of DNA abnormalities by arbitrarily primed PCR fingerprinting: allelic losses in chromosome 10q in lung cancers.

DNA fingerprinting using arbitrarily primed PCR (AP-PCR) is useful for detecting cancer-specific DNA aberrations without targeting any particular genes or knowing any nucleotide sequences in advance. AP-PCR fingerprinting is an efficient method for finding loss of anonymous chromosomal regions in cancers. We analyzed DNA from 44 human non-small cell lung cancers by fingerprinting using a single primer and found a loss of signal intensity in a DNA fragment amplified from chromosome 10 (fragment F) in 15 tumors. The detailed location of the fragment F locus on chromosome 10q was determined by PCR-based analysis of radiation hybrid panels using a sequence-tagged site established for the fragment. In 12 of the 15 tumors, loss of the signal detected by AP-PCR fingerprinting was in agreement with the results obtained by analysis of allelic imbalances using 7 polymorphic CA-microsatellite DNA markers for loci around the fragment F locus (p=0.0009). We conclude that a hitherto unknown suppressor gene for lung cancer resides at 10q in the vicinity of fragment F.

Alleles↗
PubMed1998

Use of DNA fingerprinting to detect genotoxic effects.

The effects of environmental pollutants on organisms may be monitored in a number of ways and at different levels. In the case of genotoxic chemicals, the effects on the DNA may be monitored using a number of biomarker assays capable of detecting phenotypic changes as a result of mutation, gross chromosomal abnormalities, unscheduled DNA synthesis, DNA adducts (e.g., by 32P postlabeling or by ELISA) and DNA strand breaks (e.g., by the alkaline unwinding assay or the comet assay); the sensitivity and specificity of these assays are variable. Recent developments in molecular biology such as DNA fingerprinting and gene amplification by the polymerase chain reaction (PCR) offer new possibilities for detecting DNA damage. In this laboratory, whether an alternative biomarker assay (using DNA fingerprinting by arbitrarily primed PCR) can reveal differences in the DNA fingerprints of individuals from control and polluted areas was investigated. The results indicate that DNA fingerprinting by arbitrarily primed PCR offers a useful alternative biomarker assay for detection of the genotoxic effects of environmental pollutants.

Animals↗
PubMed1991

Genetic factors accountable for line-specific DNA fingerprint bands in quail.

DNA fingerprints, prepared from mixes of DNA of individuals sampled from lines of Japanese quail selected for high or low 4-week body weight, were used to evaluate the relative contribution of several evolutionary forces to genetic diversity among populations. Comparisons between lines--two replicates of each selection direction and a control unselected line--were used to determine the frequency of line-specific DNA fingerprint bands produced by each of three major evolutionary forces: 1) mutation; 2) genetic drift; 3) selection. The latter force is expected to generate line-specific bands only if there is linkage disequilibrium between DNA fingerprint loci and quantitative loci (QTLs) controlling body weight. Using probes 33.6 and R18.1, an average of 48.4 DNA fingerprint bands in each line were analyzed. On average, 27.8 bands were found to be line-specific among the 96.8 (2 x 48.4) bands analyzed in an average comparison between pairs of lines. Based on the frequencies of line-specific bands in each particular comparison, it was calculated that 21% of the line-specific bands were due to mutation, 11% due to a single genetic drift event, 11% due to selection, 21% due to the combined effects of genetic drift and selection, 22% due to double independent events of genetic drift, and 14% due to undefined factors. Although evidence was found for a high frequency of genetic changes attributable to genetic drift, and a higher than expected frequency of linkage disequilibrium, the emphasis of this report is on the methodology suggested rather than on the particular results.

Animals↗
PubMed1991

DNA fingerprinting of the human intestinal parasite Giardia intestinalis with hypervariable minisatellite sequences.

Individual isolates of the Giardia duodenalis group of protozoan intestinal parasites were identified by DNA fingerprinting with hypervariable minisatellite sequences. A morphologically identical parasite is found in some forty different animal species. Although the species name intestinalis is reserved for the human isolates, electrophoretic karyotyping suggests that most duodenalis isolates fall into the same species grouping. Distinction based upon morphology, restriction endonuclease cleavage of genomic DNA or isoenzyme analysis has not been adequate to identify individual strains. The successful use of hypervariable sequences in the identification of individual human genomes encouraged us to examine the use of these same sequences for the possible identification of parasite isolates. We initially use as a fingerprinting probe the genome of the bacteriophage M13, which has repeated sequences recognising homologous hypervariable sequences in the human genome. The M13 probe recognises a weakly homologous set of hypervariable sequences in Giardia. The number of informative bands is comparable to those seen in mammals, since the lower molecular weight bands are also useful. There is considerable divergence in the sequences of individual Giardia minisatellites. Some cloned Giardia hypervariable sequences are more homologous to M13 than they are to each other. Similar results were observed with the hypervariable repeat sequences 3' to the human alpha-globin gene when they were used as a probe to distinguish Giardia isolates. The poly(dA-dC).poly(dG-dT) probe which recognises frequent TG tracts in a number of organisms also detects a few variable bands amidst a hybridisation background in the Giardia genome. Thus Giardia isolates which could not be distinguished by restriction endonuclease cleavage, antibody typing or isoenzyme analysis have been identified by DNA fingerprinting procedures. Detailed analysis of strain movement, resurgence, variation, host range and drug resistance is now possible. Similar families of sequences may be widespread in lower eukaryotes and useful for generating individual specific fingerprints. A procedure for detecting individual parasites is also presented. Since Giardia is regarded as the most ancient eukaryote before the occurrence of symbiosis with purple non-sulphur bacteria to generate mitochondria, the identification of hypervariable sequences in the Giardia genome should also aid in understanding the mechanism of generation and evolution of these sequences.

Animals↗
PubMed1993

DNA- and PCR-fingerprinting in fungi.

DNA-fingerprinting has been successfully used to detect hypervariable, repetitive DNA sequences (minisatellites and microsatellites) in fungi. Combined with methods used to identify random amplified polymorphic DNA (RAPD), conventional DNA-fingerprinting hybridization probes can also be used as single primers to detect DNA polymorphisms among fungal species and strains. The oligonucleotides (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, as well as the phage M13 and its core sequence, have been used as specific probes in hybridization experiments and as primers for PCR analysis. Both methods have enabled the differentiation of all the fungal species and strains that were examined, including species of Penicillium, Trichoderma, Leptosphaeria, Saccharomyces, Candida and Cryptococcus. These methods have been used 1) to clarify the taxonomic relationships among relevant species of the Trichoderma aggregate, 2) to discriminate between aggressive and non-aggressive isolates of the rape seed phytopathogen, Leptosphaeria maculans, and 3) to identify strains of the pathogenic yeasts, Cryptococcus neoformans and Candida albicans. PCR-fingerprinting allowed serotypes of C. neoformans to be distinguished. The application of DNA- and PCR-fingerprinting to fungal DNA should aid in clarification of their taxonomy and improved diagnosis of mycotic disease.

Base Sequence↗
PubMed1993

Genomic DNA fingerprints and phenotypic characteristics of serotype B Haemophilus influenzae isolates from Italy.

Three different restriction enzymes (PstI, EcoRI, SspI) were used to analyze the total genomic DNA fingerprints of 52 Haemophilus influenzae type b (Hib) isolates collected between 1982 and 1992 from patients and carriers in central-northern Italy. The same isolates were also characterized by biotyping and antimicrobial agent susceptibility typing. In addition, 13 Hib reference strains from Sweden and the Netherlands were subjected to DNA fingerprinting and compared to Italian isolates. Both genotypic and phenotypic analysis revealed low variability among the Italian study isolates. Most were biotype I and all study isolates but one were susceptible to ampicillin, chloramphenicol, rifampin, third-generation cephalosporins and cotrimoxazole. Among the 52 Italian isolates, 3 distinct DNA patterns were identified, and 88.5% of study strains belonged to the same DNA group. There was sharing of the predominant DNA profile among isolates cultured in different years from different geographical areas and different invasive, respiratory and surface infections. However, another DNA pattern was only found in carrier isolates and in one surface infection isolate. Comparison by DNA fingerprinting showed that the majority of Italian isolates were closely related to most of the analyzed Swedish and Dutch reference strains, previously shown by other techniques to be predominant in those areas. This finding provides additional support for the hypothesis that there may be a dominant European Hib clone. The results show that DNA fingerprinting is a reliable method for Hib characterization and may be a useful additional epidemiological tool for this microorganism.

DNA Fingerprinting↗
PubMed1992

DNA amplification fingerprinting of bacteria.

We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng.microliters-1 and high MgCl2 levels was especially important for reproducibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.

Animals↗
PubMed1992

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides.

DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5' terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3' terminus. Increasing annealing temperatures from 15 degrees to 70 degrees C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3'-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.

Animals↗
PubMed1995

Fluorescence-based DNA fingerprinting elucidates nosocomial transmission of phenotypically variable Pseudomonas aeruginosa in intensive care units.

DNA fingerprinting based on automated laser fluorescence analysis of randomly amplified polymorphic DNA (RAPD-ALFA) is a rapid and convenient technique for detecting clonal relatedness of bacterial isolates of nosocomial concern. During an outbreak of Pseudomonas aeruginosa among five patients in a medical intensive care unit, transmission was not suspected because of the phenotypic variability of the initial isolates. However, DNA fingerprinting by RAPD-ALFA and macrorestriction analysis identified a single genotype (strain A) for isolates from three patients and another genotype (strain B) for isolates from the remaining two patients. Strain A isolates displayed three phenotypes defined by different antibiotypes and distinct colony appearance. Retrospective analysis of DNA fingerprints demonstrated that strain A had been transmitted to the index patient one year previously in a different intensive care unit. The study demonstrates that genetic typing approaches are warranted should epidemiological relatedness be identified between phenotypically variant pathogens. Automated laser fluorescence analysis of PCR fingerprints may facilitate routine screening of bacterial isolates for in-house epidemiological surveillance. Antibiograms are an unsuitable approach for the typing of Pseudomonas aeruginosa.

Adult↗
PubMed2002

Genomic fingerprinting of virulent and avirulent strains of Clavibacter michiganensis subspecies sepedonicus.

Genomic fingerprints of C. michiganensis subsp. sepedonicus were generated by CHEF gel electrophoresis of restriction digested high-molecular weight DNA. Low levels of intra-subspecific variation were detected by cluster analysis of the fingerprints. Four haplotypes were identified by genomic fingerprinting with HindIII, and eight were identified with EcoRI. Haplotypes generated with HindIII were less similar than those generated by EcoRI. Haplotypes generated with HindIII formed groups that corresponded well with plant reactions of the strains, but similar types of groupings were less apparent with haplotypes generated with EcoRI. When disease severity in eggplant and potato, population size in potato, and ability to induce a hypersensitive response (HR) in tobacco were overlaid onto dendograms of genetic similarity, avirulent HR-negative strains clustered separately from virulent HR-positive strains in both EcoRI and HindIII profiles. Avirulent HR-positive strains that lack pCS1 clustered with avirulent HR-negative strains in a EcoRI dendogram, but clustered with virulent HR-positive strains in a HindIII dendogram. Genomic fingerprinting of high-molecular weight DNA fragments provided a means for detecting genomic variability associated with virulence in C. michiganensis subsp. sepedonicus.

Actinomycetales↗
PubMed1994

DNA fingerprint analysis of Dahl-Iwai salt-sensitive rats (S) and salt-resistant rats (R).

Using DNA fingerprint analyses, extensive molecular heterogeneity has been found between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), suggesting a doubtful value of simply comparing these rats in the study of the pathogenesis of genetic hypertension. Therefore, we evaluated the genetic similarity between Dahl salt-sensitive and salt-resistant rats newly inbred by Dr.Iwai (Dahl-Iwai S and R rats), by use of DNA fingerprint analysis. Fingerprint patterns were generated by probing Hinf I- or Alu I-digested DNA with an oligonucleotide corresponded to the tandem repeat sequence of the human myoglobin 33.6 minisatellite. These fingerprint patterns were same within each strain. S and R rts shared 82 percent of the bands in Hinf I-digested DNA and 93 percent of those in Alu I-digested DNA. Although Dahl-Iwai S and R rats are more closely related than SHR and WKY rats, multiple genetic differences still exist between these Dahl strains.

Animals↗
PubMedDate not supplied

Aspects of tuberculosis in Africa. 3. Genetic 'fingerprinting' for clues to the pathogenesis of tuberculosis.

The recent discovery of a repetitive element within the DNA of Mycobacterium tuberculosis, which is present in variable numbers at different locations in separate strains of the organism, has led to the development of genetic 'fingerprinting' to distinguish between different isolates. Clusters of cases of tuberculosis have been identified in Europe and the USA in which the organisms cultured had identical 'fingerprints' confirming that transmission was occurring. Unrelated isolates generally have distinct 'fingerprints'. In Africa, where transmission is more common than in Europe, there is less heterogeneity between isolates. We have typed 117 isolates of M. tuberculosis collected from continuing studies in Malawi and Kenya. Paired isolates from an individual patient produced matching 'fingerprints' in 22 of 25 cases. There were 18 isolates which had an identical matched pair from a separate patient; we have not yet found any epidemiological link between these patients. These data show that there is sufficient heterogeneity amongst African isolates of M. tuberculosis to make studies of transmission feasible and to address questions of pathogenesis and epidemiology.

DNA Fingerprinting↗
PubMed1992

Does chemotherapy of hematological malignancies affect DNA fingerprint pattern?

We analyzed DNA fingerprints of lymphoma patients to find out whether DNA damage caused by irradiation and chemotherapy can result in DNA fingerprint changes, and whether the differences found previously in leukemia patients could be partially due to the treatment. In this study we did not find any post-treatment DNA fingerprint differences in 33 lymphoma patients, concluding, that the therapy of hematological malignancies does not affect DNA fingerprint patterns. Further, the variations of methylation do not either explain the detected differences in leukemic patients.

DNA Damage↗
PubMed1992

Detection of DNA alterations in human bladder tumors by DNA fingerprint analyses.

DNA fingerprint analyses were used to examine the constitutional and tumor DNA from 22 bladder tumor patients. DNA alterations, such as loss of bands, new bands, and intensity shifts were observed in 10 of the 22 patients. The most frequent DNA alteration, occurring in 80% of the patients, was a complete loss of one or several bands. Fingerprint abnormalities were present both in low-malignant superficial tumors and in high-malignant invasive tumors, but were also lacking in the latter group. Apparently no relationship exists between fingerprint abnormalities and gross chromosomal aberrations or the proportion of S-phase cells as measured by flow cytometry or development of recurrent tumors during a limited observation period. Thus, whether fingerprint aberrations express genetic alterations directly involved in the malignancy potential of bladder carcinoma remains an open question.

Adult↗
PubMedDate not supplied

The use of biotyping and DNA fingerprinting in typing Candida albicans from hospitalized patients.

The application of typing procedures for the purpose of strain differentiation among isolates of Candida albicans obtained from hospitalized patients has been limited. We have applied biotyping and DNA restriction fragment analysis (DNA fingerprinting) by using EcoRI to the study of C. albicans isolates obtained from hospitalized patients. A total of 68 isolates from 15 patients were studied. Thirteen subtypes were identified by biotyping, 8 by DNA fingerprinting, and 21 by a combination of the biotyping and DNA fingerprinting approaches (composite subtype). Both techniques were highly reproducible. In examining the strain variation among isolates obtained from multiple anatomic sites over time, we found that similar, if not identical, strains were recovered from the oropharynx, urine, stool, and blood in a given patient, and these strains persisted. Only rarely did two patients share the same composite subtype suggesting sporadic nosocomial transmission. The combination of biotyping and DNA fingerprinting improved strain discrimination compared to either method alone. Further investigation with these and other epidemiologic typing methods will be necessary to enhance the understanding of the epidemiology and pathogenesis of candidiasis in hospitalized patients.

Bacterial Typing Techniques↗
PubMed1995

The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints.

Pathogenic Vibrio cholerae strains were compared by fingerprinting with arbitrarily primed polymerase chain reaction (AP-PCR). They were O1 classical and El Tor strains and recent non-O1 Bengal strains. Ten oligonucleotides from a total of fifty-two tested gave distinctive patterns, and these strains were separated into four groups. A second technique, amplification of 16S/23S rRNA spacers with a pair of oligonucleotides, was also used. Various bands were obtained, and the result can be treated as an additional fingerprint with a different pattern for each of the groups. The method of AP-PCR fingerprinting is fast and sensitive. A test of the stability of the El Tor patterns was done with a set of strains isolated during the present Brazilian epidemics. Examples of AP-PCRs with non-O1 strains are given. A typing scheme is proposed in which oligo 1 is first used, and depending on the fingerprint obtained, additional oligonucleotides are used to confirm the classification of the strain. It is proposed that the AP-PCR technique be used for epidemiological studies, analysing strains reaching new locations or environmental isolates suspected of being pathogenic. It will be particularly helpful in cases in which traditional methods cannot clearly classify the strain.

DNA Fingerprinting↗
PubMed2005

Fingerprinting of single viral genomes.

We demonstrate the use of technology developed for optical mapping to acquire DNA fingerprints from single genomes for the purpose of discrimination and identification of bacteria and viruses. Single genome fingerprinting (SGF) provides not only the size but also the order of the restriction fragments, which adds another dimension to the information that can be used for discrimination. Analysis of single organisms may eliminate the need to culture cells and thereby significantly reduce analysis time. In addition, samples containing mixtures of several organisms can be analyzed. For analysis, cells are embedded in an agarose matrix, lysed, and processed to yield intact DNA. The DNA is then deposited on a derivatized glass substrate. The elongated genome is digested with a restriction enzyme and stained with the intercalating dye YOYO-1. DNA is then quantitatively imaged with a fluorescence microscope and the fragments are sized to an accuracy >or=90% by their fluorescence intensity and contour length. Single genome fingerprints were obtained from pure samples of adenovirus, from bacteriophages lambda and T4 GT7, and from a mixture of the three viral genomes. SGF will enable the fingerprinting of uncultured and unamplified samples and allow rapid identification of microorganisms with applications in forensics, medicine, public health, and environmental microbiology.

Calibration↗