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IS6110 DNA fingerprinting analysis of individually separated colonies of Mycobacterium tuberculosis.

There are no data so far that show IS6110 restriction fragment length polymorphism (RFLP) patterns of individually separated tuberculosis bacilli from clinical isolates, and their alterations during follow-up surveys. We picked 20-60 tuberculosis clones from clinical isolates under anti-tuberculosis medication, and individually analysed their DNA fingerprinting patterns using IS6110 RFLP as well as spoligotyping as a second typing. The study using cloned bacilli of Mycobacterium tuberculosis showed that clinical isolates contained several clones with different DNA fingerprints and that their band patterns altered weakly but distinctly during follow-up surveys. However, there was no significant difference in the fingerprinting patterns when clinical isolates were to RFLP without separating to subjected/individual colonies. In view of the IS6110 RFLP of individually separated tuberculosis bacilli, we have now speculated several possibilities: (1) that clones with different DNA fingerprints exist in clinical isolates; (2) that IS6110 RFLP patterns of the materials depend on the population of the original clone and the variants having DNA fingerprints different from the original pattern; and (3) that their band patterns are influenced not only by the stability of the original germ having its own fingerprint, but also by the fragility of the new clones.

Adult↗

[Transmission of sensitive and resistant strains of Mycobacterium tuberculosis in The Netherlands, 1993-1995, studied by means of DNA fingerprinting].

OBJECTIVE: Determination of the contribution of recent transmission of both sensitive and drug-resistant Mycobacterium tuberculosis strains to the number of tuberculosis cases in the Netherlands. DESIGN: Descriptive study. SETTING: National Institute for Public Health and Environment, Bilthoven, the Netherlands. METHODS: Since 1993 all isolates of M. tuberculosis in the Netherlands are sent to the national reference laboratory for surveillance purposes. The strains with IS6110 DNA fingerprint, isolated from January 1993 to July 1995, were analysed for clustering of DNA patterns (clustering of identical DNA patterns was assumed to represent recent transmission of tuberculosis). A transmission index was calculated from the ratio of the number recently infected tuberculosis patients and the number of source patients. RESULTS: Among 2,217 M. tuberculosis isolates, 1,313 unique DNA fingerprints were observed, while 264 DNA patterns occurred more than once. 904 (41%) DNA fingerprints were part of a cluster of identical fingerprints. The mean cluster size was 3.42. The 232 resistant strains showed significantly less clustering (33% versus 42%, p < 0.02) and a smaller transmission index (0.27 versus 0.42, p < 0.02) compared with sensitive strains. CONCLUSION: Recent transmission contributes to the magnitude of the tuberculosis problem in the Netherlands. The epidemiological situation would, however, lead to gradual elimination of the disease were it not for introduction of tuberculosis from other countries. Transmission of resistant strains is relatively limited. Micro-epidemics caused by resistant M. tuberculosis strains were not observed.

DNA Fingerprinting↗

Individualization and estimation of relatedness in crocodilians by DNA fingerprinting with a Bkm-derived probe.

Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22-23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.

Alligators and Crocodiles↗

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA.

We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3' termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I from Escherichia coli, which has 3'-->5' exonuclease and replacement activities as well as its main 5'-->3' polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.

Animals↗

Assessment of DNA fingerprinting for rapid identification of outbreaks of systemic candidiasis.

DNA fingerprinting was assessed as an improved typing system for Candida albicans aimed at speeding the implementation of cross infection control measures in outbreaks of systemic candidiasis. The study was carried out with 45 previously characterised isolates from five different outbreaks and with 96 unrelated isolates from a mixed control population. Sixteen different genotypes were produced. Results were obtainable within days, reproducibility was high, and there was good discrimination among different outbreaks. Compared with existing typing systems DNA fingerprinting provides a robust system that may be used rapidly to identify outbreaks of nosocomial candidiasis in laboratories with no specialist skill in typing C albicans.

Candida albicans↗

Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans.

In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms.

Base Sequence↗

Positive identification of an immigration test-case using human DNA fingerprints.

The human genome contains a set of minisatellites, each of which consists of tandem repeats of a DNA segment containing the 'core' sequence, a putative recombination signal in human DNA. Multiallelic variation in the number of tandem repeats occurs at many of these minisatellite loci. Hybridization probes consisting of tandem repeats of the core sequence detect many hypervariable minisatellites simultaneously in human DNA, to produce a DNA fingerprint that is completely individual-specific and shows somatic and germline stability. These DNA fingerprints are derived from a large number of highly informative dispersed autosomal loci and are suitable for linkage analysis in man, and for individual identification in, for example, forensic science and paternity testing. They can also be used to resolve immigration disputes arising from lack of proof of family relationships. To illustrate the potential for positive or inclusive identification, we now describe the DNA fingerprint analysis of an immigration case, the resolution of which would have been very difficult and laborious using currently available single-locus genetic markers.

DNA, Satellite↗

DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica.

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).

Crosses, Genetic↗

Construction and use of a computerized DNA fingerprint database for lactic acid bacteria from silage.

Efficient selection of new silage inoculant strains from a collection of over 10,000 isolates of lactic acid bacteria (LAB) requires excellent strain discrimination. Toward that end, we constructed a GelCompar II database of DNA fingerprint patterns of ethidium bromide-stained EcoRI fragments of total LAB DNA separated by conventional agarose gel electrophoresis. We found that the total DNA patterns were strain-specific; 56/60 American Type Culture Collection strains of 33 species of LAB could be distinguished. Enterococcus faecium strains ATCC19434 and ATCC35667 had identical total DNA patterns and RiboPrints. Lactobacillus rhamnosus strains ATCC7469 and ATCC27773 also had identical total DNA patterns, but different RiboPrints. EcoRI RiboPrint patterns could distinguish only about 9/23 Lactobacillus plantarum strains and about 6/10 Lactobacillus buchneri strains, whereas all 33 strains could be distinguished by EcoRI total DNA patterns. Despite gel-to-gel variation, new DNA patterns can be readily grouped with existing patterns using GelCompar II. The database contains large homogenous clusters of L. plantarum, E. faecium, L. buchneri, Lactobacillus brevis and Pediococcus species that can be used for tentative taxonomic assignment. We routinely use the DNA fingerprint database to identify and characterize new strains, eliminate duplicate isolates and for quality control of inoculant product strains. The GelCompar II database has been in continuous use for 7 years and contains more than 3600 patterns representing approximately 700 unique patterns from over 300 gels and is the largest computerized DNA fingerprint database for LAB yet reported.

DNA Fingerprinting↗

Cloning and characterization of a complex DNA fingerprinting probe for Candida parapsilosis.

Candida parapsilosis accounts for a significant number of nosocomial fungemias, but in fact, no effective and verified genetic fingerprinting method has emerged for assessing the relatedness of independent isolates for epidemiological studies. A complex 15-kb DNA fingerprinting probe, Cp3-13, was therefore isolated from a library of C. parapsilosis genomic DNA fragments. The efficacy of Cp3-13 for DNA fingerprinting was verified by a comparison of its clustering capacity with those of randomly amplified polymorphic DNA analysis and internally transcribed spacer region sequencing, by testing species specificity, and by assessing its capacity to identify microevolutionary changes both in vitro and in vivo. Southern blot hybridization of EcoRI/SalI-digested DNA with Cp3-13 provides a fingerprinting system that (i) identifies the same strain in independent isolates, (ii) discriminates between unrelated isolates, (iii) separates independent isolates into valid groups in a dendrogram, (iv) identifies microevolution in infecting populations, and (v) is amenable to automatic computer-assisted DNA fingerprint analysis. This probe is now available for epidemiological studies.

Candida↗

Usefulness of the secondary probe pTBN12 in DNA fingerprinting of Mycobacterium tuberculosis.

A comparison was made between DNA fingerprints of Mycobacterium tuberculosis produced with the insertion sequence IS6110 and those produced with the polymorphic GC-rich repetitive sequence contained in the plasmid pTBN12. A total of 302 M. tuberculosis isolates from the prison system in Madrid, Spain, and the Denver Public Health Department (Denver, Colo.) were analyzed with the two probes. Both probes identified the same isolates in the same clusters when the fingerprints had six or more copies of IS6110. Analysis of isolates with unique IS6110 fingerprints demonstrated that they were unique with pTBN12. The pTBN12 probe had greater discriminating power in isolates having five or fewer copies of IS6110. Forty-seven isolates from Denver having fewer than five copies of IS6110 which were grouped in 11 clusters with identical fingerprint patterns were subdivided into 35 different patterns by pTBN12. Isolates with IS6110 fingerprints with more than six copies of IS6110 that differed from one another by only one or two hybridizing bands were analyzed with pTBN12. Most of these sets of isolates demonstrated identical patterns with pTBN12. However, some exceptions were observed, suggesting that those having nearly identical IS6110 patterns should not necessarily be included in the same cluster. Since IS6110 provides more polymorphism in the fingerprint, it is most useful in identifying isolates with unique fingerprint patterns and those in clusters in which the isolates contain six or more copies of the insertion. However, it is necessary to employ a secondary probe, such as pTBN12, to discriminate isolates with five or fewer copies of IS6110 and those with similar but not identical IS6110 patterns.

DNA Fingerprinting↗

Extra-pair sires as identified by means of standardized across-gel comparisons of multilocus DNA fingerprints.

Molecular analyses of genetic parentage in wild bird populations demonstrate that extra-pair paternity (EPP) is common, even in socially monogamous species. After having identified extra-pair offspring (EPO), an important step is to uncover the identity of extra-pair sires (EPS). Studies relying on multilocus DNA fingerprinting face the problem that simple between-gel comparisons of fingerprint banding patterns are imprecise in assigning parentage across gels. To tackle this problem, we developed a method to identify EPS by means of standardized across-gel comparisons of multilocus DNA fingerprints and applied it in a socially monogamous bird species with high levels of EPP, the coal tit (Parus ater). Scanned DNA fingerprint gels and standard image-editing software allowed to screen and reliably identify EPS. Overall, EPP was successfully assigned to 32 different EPS for 143 out of 210 (68.1%) EPO detected. This corresponds well to extra-pair parentage assignment rates revealed by microsatellite-based studies in congeneric species. Our approach proves especially useful whenever multilocus DNA fingerprints for parentage exclusion exist while microsatellite markers are not available.

Animals↗

Simultaneous DNA 'fingerprinting', diagnosis of sex and single-gene defect status from single cells.

Sex and cystic fibrosis status have been previously diagnosed separately at the single cell level. We have developed a sensitive, reliable, accurate and rapid (within 5-6 h) system for the simultaneous diagnosis of sex, cystic fibrosis and a DNA 'fingerprint' within a single reaction from a variety of single cells. As contamination cannot be totally excluded, particularly at the single cell level, DNA 'fingerprinting' can be used to assess the risk of contamination. High sensitivity with single cells is combined with very high specificity (estimated matching probability of 10(-7)-10(-8)), allowing the source of the amplified cell to be identified with a very high degree of probability. Fluorescent primers were multiplexed for six tetranucleotide microsatellite sequences to determine the DNA fingerprint; the amelogenin gene was used to diagnose sex, and primers for the CFTR region were used to determine cystic fibrosis (CF) status. Analysis of the fluorescent product was undertaken using an automated DNA sequencer with Genescan software. This technique has many applications such as prenatal and preimplantation diagnosis, forensic identification of small or degraded samples, and detection of contamination sources. DNA fingerprints of single haploid spermatozoa and other cells can be assessed, so ensuring the detection of both diploid and haploid contamination during preimplantation diagnosis.

Cystic Fibrosis↗

DNA fingerprinting of Listeria monocytogenes using enterobacterial repetitive intergenic consensus (ERIC) motifs-polymerase chain reaction/capillary electrophoresis.

The molecular technique, enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) produces genomic DNA fingerprint that discriminate bacterial species and strains. This technique was applied to the characterization of Listeria monocytogenes, an important food-borne pathogen implicated in numerous cases of listeriosis. The ERIC-PCR resulted in distinct DNA fingerprinting patterns of all L. monocytogene serotypes and Listeria species. Analysis of the genomic DNA fingerprints was accomplished using capillary electrophoresis (CE), an alternative technique to the conventional agarose gel method. The optimization of CE conditions (electrokinetic injection, applied voltage) resulted in the resolution of amplified DNA fragments up to 1000 bp. Comparisons of electropherograms provided genomic fingerprint templates which could be further used for supplementary information. The ERIC-PCR method coupled to CE provides a rapid technique in differentiating bacterial spp., and may contribute relevant information in food-borne outbreak studies.

DNA Fingerprinting↗

Paternity testing of endangered species of birds by DNA fingerprinting with non-radioactive labelled oligonucleotide probes.

In the last years, DNA fingerprinting became the most powerful tool for identification and paternity testing in man. The success of this method encouraged the German Federal Ministry of Environment, Natural Protection and Reactor Safety to apply DNA fingerprinting in the field of protection of endangered species of birds, such as birds of prey or parrots. In the last three years, we received more than 400 blood and tissue samples of 23 species of birds of prey or parrots, most of them obtained by confiscation, to establish paternity and legal breeding success. We used digoxigenated oligonucleotide probes, mainly (GGAT)4 and (GACA)4 for hybridization. In most cases of confiscated families of birds, paternity testing showed exclusions of nestlings.

Animal Husbandry↗

DNA fingerprint analysis in chemically mutagenized Chinese hamster lung cells.

Using a multi-locus minisatellite Per-6 DNA probe, we performed DNA fingerprint analysis. Chinese hamster lung (CHL) cells were treated with six model chemicals: N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methyl methanesulfonate, furylfuramide, 2-acetylamino-fluorene, and cyclophosphamide, with or without S9 mix. 771 hypoxanthine phosphoribosyltransferase deficient clones (749 from mutagen-treated cells and 22 from untreated cells) and 90 unselected clones from untreated cells were isolated and analyzed. The spontaneous mutation frequency at CHL cell minisatellite loci was 0.31-0.63%. All the chemicals increased mutation frequencies. Almost all mutations localized to the three specific minisatellite loci corresponding to 4.2, 3.8, and 2.4 kb bands, suggesting that these regions are more unstable and susceptible to mutation. DNA fingerprint analysis is a promising technique for detecting mutations at neutral DNA regions, especially recombinational mutations, and may be useful for surveying genetic instability related to heritable defects or aging.

2-Acetylaminofluorene↗

Multilocus DNA fingerprinting and RAPD reveal similar genetic relationships between strains of Oreochromis niloticus (Pisces: Cichlidae).

Two molecular techniques which reveal highly variable DNA polymorphisms, RAPD and multilocus DNA fingerprinting, were used to evaluate genetic diversity between six aquacultural strains of Oreochromis niloticus (tilapia) from the Philippines. The results using both techniques were in close agreement. Within-strain heterozygosity values were similar and were correlated between the two data sets, but statistical errors associated with the RAPD data set were lower. Although genetic distances between strains were greater using DNA fingerprinting, the distances measured using both methods were significantly correlated. Both methods were useful in estimating variation between strains, but they offered different advantages. RAPD was technically easier to perform and produced results with low statistical error, whereas DNA fingerprinting detected greater genetic differentiation between strains. The theoretical basis for using RAPD and multilocus minisatellite markers for population studies is discussed.

Animals↗

[DNA diagnosis in veterinary medicine: I. DNA fingerprinting].

With the development of recombinant DNA-technology during the last 15 years, it became possible to analyse genetic variability directly at the level of genomic DNA. Restriction-Fragment-Length-Polymorphism (RFLP) and Highly-Variable-Regions (HVRs) lead to the development of new genetic markers, which can be used for the localization of normal and mutated genes. Some polymorphisms are hypervariable and can therefore be applied to carry out DNA-fingerprints thus identifying individuals from man and animals.

Animals↗