DNA Fingerprint Profiles Research Articles

Intra-species differentiation of Trypanosoma evansi by DNA fingerprinting with arbitrary primered polymerase chain reaction

A method was developed to obtain reproducible DNA fingerprints from isolates of Trypanosoma evansi by PCR-based amplification using arbitrary primers (AP-PCR). Only one out of 10 randomly designed 12-mer primers generated DNA fingerprint profiles that revealed intra-species differences in T. evansi. The technique was applied in association with parasitological and serological examinations to investigate animal-to-animal transmission during an outbreak of surra in Thailand. The AP-PCR method has the advantage of being simple, fast and sensitive to diagnosis and characterization of the parasites since it does not require prior DNA sequence information. The technique should prove useful for the proper understanding of epidemiology and for designing rational control programs for trypanosomosis.

Optimization of the arbitrarily-primed polymerase chain reaction (AP-PCR) for intra-species differentiation of Vibrio vulnificus

Optimal parameters were established for obtaining unique and reproducible DNA fingerprints of selected clinical and environmental isolates of Vibrio vulnificus by the arbitrarily primed polymerase chain reaction (AP-PCR). Genomic DNA from selected strains was subjected to AP-PCR amplification using a single, arbitrarily selected oligonucleotide primer, R-PSE420. Amplified DNA was analyzed by agarose gel electrophoresis and ProRFLP® computer software. Reproducibility of the fingerprint was dependent upon the concentrations of the purified genomic DNA, MgCl 2 and oligonucleotide primer, and on PCR cycling parameters. Using 1 μg of purified genomic DNA, 2.5 mM MgCl 2, 1.04 μM of R-PSE420 oligonucleotide primer and thermal cycling protocols with stepwise increases in the annealing temperatures, DNA fingerprints which were reproducible and free of primer artifacts were generated. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each V. vulnificus strain tested were produced. These AP-PCR generated unique DNA fingerprint profiles can be used in the identification, and investigation of the distribution and diversity of various strains of V. vulnificus in bivalve shellfish and their surrounding waters.

Measuring heterogeneity in forensic databases using hierarchical Bayes models

SUMMARY As currently defined, DNA fingerprint profiles do not uniquely identify individuals. For criminal cases involving DNA evidence, forensic scientists evaluate the conditional probability that an unknown, but distinct, individual matches the crime sample, given that the defendant matches. Estimates of the conditional probability of observing matching profiles are based on reference populations maintained by forensic testing laboratories. Each of these databases is heterogeneous, being composed of subpopulations of different heritages. This heterogeneity has an impact on the weight of the evidence. A hierarchical Bayes model is formulated that incorporates the key physical characteristics inherent in these data. With the help of Markov chain Monte Carlo sampling, levels of heterogeneity are estimated for three major ethnic groups in the database of Lifecodes Corporation.

Brazilian isolates of Trypanosoma cruzi from humans and triatomines classified into two lineages using mini-exon and ribosomal RNA sequences.

Traditional molecular and biochemical methods, such as schizodeme analysis, karyotyping, DNA fingerprinting, and enzyme electrophoretic profiles, have shown a large variability among Trypanosoma cruzi isolates. In contrast to those results, polymerase chain reaction (PCR) amplification of sequences from the 24S alpha ribosomal RNA gene and from the mini-exon gene nontranscribed spacer indicated a dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 86 T. cruzi field stocks (68 isolated from humans with defined presentations of Chagas' disease and 18 from triatomines) derived from four Brazilian geographic areas were typed by the PCR assay based on the DNA sequences of the mini-exon and 24S alpha rRNA genes. These stocks were ordered into the two major T. cruzi lineages. Lineage 1 was associated mainly with human isolates and lineage 2 with the sylvatic cycle of the parasite.

Subtyping of Streptococcus dysgalactiae and Streptococcus uberis isolated from bovine mammary secretions by DNA fingerprinting.

Streptococcus dysgalactiae and Streptococcus uberis isolated from mammary secretions of cows from Tennessee and New Zealand were subtyped using polymerase chain reaction-based DNA fingerprinting. Such DNA fingerprinting using primer 8.6d (5'-GTAACGCC3') resulted in categorizing 116 S. dysgalactiae isolates into 25 different subtypes, with 17 subtypes observed in isolates from Tennessee and eight in isolates from New Zealand. All S. dysgalactiae DNA fingerprint profiles, regardless of origin, contained 700- and 330-base pair fragments. The majority of S. dysgalactiae isolates (73%) from Tennessee belonged to two subtypes. The remaining 23 isolates belonged to 15 different DNA fingerprint subtypes. Streptococcus dysgalactiae isolates from New Zealand (n = 32) were grouped into eight different subtypes; 66% belonged to two subtypes. A characteristic feature of S. dysgalactiae isolates from New Zealand was the presence of a 270-base pair DNA fragment seen infrequently in S. dysgalactiae isolates from Tennessee. When primer OPE-4 (5'-GTGACATGCC-3') was used, DNA fingerprinting differentiated S. uberis from Tennessee (n = 28) and New Zealand (n = 30) into 20 subtypes; 14 subtypes were observed in isolates from Tennessee, and six in isolates from New Zealand. All S. uberis DNA fingerprint profiles, regardless of origin, contained 1100-, 640-, and 450-base pair fragments. A characteristic feature of S. uberis isolates from New Zealand was the presence of a 300-base pair DNA fragment seen infrequently in S. uberis isolates from Tennessee. The most common subtypes of S. dysgalactiae and S. uberis from Tennessee herds were isolated in milk from lactating cows during monthly herd surveys, in milk from cows with clinical mastitis, and in mammary secretions from cows during the periparturient period, and thus were not confined to one particular stage of lactation. These data suggest that S. dysgalactiae and S. uberis from New Zealand are distinct from those isolated from the USA, and that DNA fingerprinting can be used as an epidemiological tool to differentiate streptococci and identify important sources of these mastitis pathogens on dairy farms.

DNA Fingerprinting of Riemerella anatipestifer

Seventeen restriction endonucleases were evaluated for use in DNA fingerprinting of Riemerella anatipestifer. Digestion of chromosomal DNA with either HinfI or DdeI restriction endonuclease, followed by submarine electrophoresis in agarose and staining with ethidium bromide, resulted in DNA fingerprint profiles that could be easily resolved. HinfI produced readable fingerprint patterns in the 2.7-20-kb range and was used to distinguish DNA fingerprint profiles among 89 strains of R. anatipestifer representing isolates from various avian species in the United States, the United Kingdom, Australia, Canada, Germany, and Israel. A total of 52 distinct DNA fingerprint profiles were found.

Preparation and use of alkaline-phosphatase-conjugated oligonucleotide probes for single-locus and multilocus VNTR analysis.

Nonisotopic probes have been widely adopted for DNA fingerprinting and DNA profiling because of their ease and speed of use and obvious safety and environmental advantages ().

A Simple and Rapid Procedure for Isolation of Total DNA Suitable for Fingerprint Analysis from shape Amaranthus

A simple, efficient and reliable method is described for isolation of total DNA from young leaves of Amaranthus species. This procedure yields a high amount (600–800 µg DNA/g fresh leaf tissue) of good quality DNA free from contaminating proteins, polysaccharides, and coloured pigments. The DNA is suitable for digestion with several restriction endonucleases, preparation of Southern blots, and PCR amplification. The DNA has been successfully used for generating DNA fingerprint profiles and RAPD banding patterns in two species of Amaranthus. The procedure is suitable for processing of a large number of samples simultaneously.

Multilocus DNA fingerprints in the plant Cynoglossum officinale L. and their use in the estimation of selfing

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale. Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four‐base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding ‘between‐gel’ comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing.

Spontaneous and induced minisatellite instability.

Minisatellites provide not only the basis for DNA fingerprinting and DNA profiling but also extremely informative systems for analysing processes of tandem repeat turnover in the human genome. Minisatellite instability appears to involve distinct mutation processes in somatic and germline cells; in the germline, mutation is frequently dominated by inter-allelic conversion-like events most likely occurring at meiosis and apparently regulated by cis-acting mutation initiator elements. Attempts to define these initiators in transgenic mice have so far been thwarted by what appears to be a major human/mouse barrier to the inter-species transfer of repeat instability. Minisatellites not only show high frequency spontaneous mutation in the germline, but also appear to be very sensitive to mutation induction by ionizing radiation, both in experimentally irradiated mice and in human populations exposed following the Chernobyl disaster; the mechanisms of mutation induction by radiation remain enigmatic.

Evaluation of the genetic diversity among maize lines (Zea mays L.) by DNA fingerprinting analysis

Informativo DNA fingerprint profiles of eight homozygotic maize lines were obtained by the electrophoretic separation of DNA restriction fragments and the ir hybridization with the minisatellite probe R18.1. The analysis of the bandsharing frequencies allowed to identify all the lines and to estimate the genetic distances between them. The relationship obtained by DNA fingerprinting analysis of the eight inbreed lines was highly consistem with the ir genetical origin.

Rearrangements at a DNA-fingerprint locus in the rice blast fungus.

Populations of the rice blast fungus, Magnaporthe grisea, can be sorted into clonal lineages based on restriction fragment length polymorphisms (RFLPs) detected with the repetitive DNA sequence MGR586. Mechanisms that produce DNA-fingerprint variation among lineages, are not known. In the process of analyzing the meiotic segregation of MGR586 RFLPs we identified a novel polymorphism, called MGR586-P2, in one member of a sister-spore pair from a complete tetrad. Molecular cloning revealed that P2 was generated by a nearby insertion of a novel, long-terminal-repeat (LTR)-containing retrotransposon. Genetic analysis showed that P2 and its progenitor polymorphism (P1) are alleles of a single polymorphic locus termed MGR586-PL (PL). Surprisingly, we found that a strain harboring PL recurrently produced clonal descendants harboring P1, P2 and possibly a third allele designated P3. PL is not located near a telomeric region. Our results show that some DNA-fingerprint loci may be hypervariable and undergo recurrent rearrangements. These findings have implications for interpreting DNA-fingerprint profiles from M. grisea populations.

MICROGEOGRAPHIC PATTERNS OF GENETIC AND MORPHOLOGICAL VARIATION IN SAVANNAH SPARROWS (PASSERCULUS SANDWICHENSIS).

Surveys of genetic population structure are often limited to large geographic scales because geographically close populations are indistinguishable. Genetic uniformity across adjacent demes can be interpreted as evidence for cohesion (panmixia) or recent divergence. However, poor genetic resolution at microgeographic scales can also arise from the use of overly conservative (slowly evolving) markers. This study examines the ability of hypervariable, minisatellite loci to discriminate among geographically close populations of Savannah sparrows (Passerculus sandwichensis) and to track morphological differentiation at a microgeographic scale (interregional distance < 55 km). Savannah sparrows breeding at five island and two mainland sites in the Bay of Fundy, New Brunswick, Canada, show concordant patterns of variation in external morphology (seven characters) and multilocus DNA fingerprinting profiles (Sxy ): island sparrows are phenotypically larger and genetically more similar to each other than they are to mainland sparrows. This pattern of variation is consistent with both adaptive (natural selection) and nonadaptive (genetic drift) mechanisms of population divergence. Based on minisatellite diversity, the effective size of both island and mainland populations is 37, an estimate substantially lower than census population sizes. These data are discordant with observations of sparrow vagility and abundance and suggest a closer examination of microgeographic patterns in avian systems.

Characterization and population diversity of interspersed repeat sequence variants (IRS-morphs)

Inter-Alu PCR is increasingly useful in human genome mapping studies. One use is the generation of alumorphs, polymorphisms resulting from the presence or absence of inter-Alu PCR products. In this study, we have increased the proportion of the genome that can be analyzed by this technique with the use of long interspersed elements (LINEs). The set of polymorphisms detected by both Alu and LINE primers are referred to as interspersed repetitive sequence variants or IRS-morphs. Since a presence-absence variant may have been the result of a recent Alu or LINE insertion, we analyzed 7 isolated IRS-morphs that were generated, in part, with a primer derived from either a consensus LINE or a young Alu subfamily specific sequence, and observed by Southern blot analysis that these variants resulted from other types of genomic alterations. The use of these primers, however, reduces background from the numerous LINEs and Alu elements in the genome, providing sharp DNA fingerprint profiles. We have demonstrated the potential usefulness of these IRS-morph profiles in human population studies. We compared 12 IRS-morphs from a single amplification reaction from five distinct population groups (Caucasian (northern European descent), Hispanic (Mexican-American), Hindu-Indian, Papua New Guinean, and Greenland Eskimo) and observed that most have variable allelic frequencies among populations. The utilization of additional IRS-morph profiles will perpetuate this technique as a tool for DNA fingerprinting and for the analysis of human populations. Key words : Alu elements, DNA fingerprint, human populations, LINEs, SINEs.

An evaluation of new gel matrices for the separation of PCR-amplified DNA fragments.

It has been shown that the novel gel matrix, PCR Purity Plus which consists of a vinyl-polymer of polyacrylamide, provides a superior and rapid means of separating DNA fragments. As this product has been discontinued, the two new commercial versions of this matrix (MDE and GeneAmp) with PCR Purity Plus were compared. Optimal conditions for resolving DNA fingerprint profiles for both matrices were defined. Both MDE and GeneAmp gels provided a clear separation of DNA fragments. However, the profiles obtained on GeneAmp gel were closest to that of PCR Purity Plus. These results should be useful to DNA fingerprinting studies where it is critical to obtain a clear resolution of complex DNA profiles.

Cloning of a DNA repeat element from horse: DNA sequence and chromosomal localization.

A DNA repeat element, revealed initially by digestion of horse DNA with TaqI, was cloned and characterized by Southern and in situ hybridization studies and nucleotide sequencing. The clone, e4/1, consisted of 32 tandem reiteration of a unit repeat of 21-22 bp, and produced multilocus DNA fingerprinting profiles that were useful for parentage analysis in horses. The tandem repeat element was shown by in situ hybridization to be localized in the centromeres of the acrocentric but not metacentric classes of horse chromosomes.

An epidemiological study of Candida species infection in cancer patients using genetic fingerprinting and morphotyping

During a six-week period a cluster of four cases of Candida fungaemia occurred in a mixed haematology/oncology unit of a large Dublin teaching hospital. A study was conducted to determine whether the cluster of cases was caused by a particular strain. Nine patients were studied; five who were colonized with Candida spp. and four who developed Candida fungaemia. Twenty-two clinical isolates of Candida spp. were collected and identified. Three of the patients with fungaemia yielded Candida albicans from blood cultures and C. tropicalis was isolated from the fourth patient. C. albicans isolates were serotyped, morphotyped and analysed by DNA fingerprinting of total cellular DNA using the cloned C. albicans-specific, mid-repeat sequence element 27A as a molecular probe. All C. albicans isolates were of serotype A. Eight distinguishable types were identified by both morphotyping and DNA typing from 19 C. albicans isolates recovered from seven individual patients, although there were several discrepancies. Of three patients from whom two or more isolates of C. albicans were recovered on separate occasions, two yielded recurrent isolates with different morphotype codes. However, in both cases, the recurrent isolates from individual patients yielded indistinguishable, or closely related, DNA fingerprint profiles. Both morphotyping and DNA fingerprint analysis readily distinguished the three blood culture isolates of C. albicans. We conclude that the Candida spp. infections in the unit were not due to cross-infection and were probably related to the patients' indigenous flora.

Oncogenic transformation and growth factor production of a human urothelial cell line

The objective of this study was to demonstrate that a nontumorigenic simian virus 40 (SV40) immortalized human urothelial cell line can be tumorigenically transformed following growth in a serum free, growth factor free, chemically defined culture system. The tumorigenicity of the cells was determined by the generation of tumors after inoculation into athymic nude mice. A cell line was derived from one of the tumors and the origin of the transformed cells was confirmed by immunohistochemical staining and analysis of the DNA fingerprint profile. Conditioned medium derived from the nontumorigenic parent cell line and the tumor line contained an activity that synergized with recombinant granulocytemacrophage colony stimulating factor (rmu GM-CSF) and recombinant macrophage colony stimulating factor (rhu M-CSF) to produce large colonies in bone marrow cultures (HPP-CFC). The concentration of human stem cell factor (SCF) and human Interleukin-I a-like activity produced by the urothelial cells was lower in the medium conditioned by the transformed tumorigenic cells than by the nontumorigenic parental cells. This data suggest that there are differences in the growth factor production of normal urothelial and tumor cells. It is possible that tumors deprived of normal levels of growth factors and nutrients may undergo further tumor progression, thus contributing to the heterogeneity seen in the clinic.

Identification and Strain Differentiation of 'Bacteroides fragilis Group' Species and Prevotella bivia by PCR Fingerprinting

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.

DNA Fingerprint Analysis of Standard, Intermediate, and Variant Antigenic Types of Salmonella enterica Subspecies enterica Serovar Gallinarum Biovar pullorum

DNA fingerprint profiles and antigenic types of 150 Salmonella enterica serovar Gallinarum biovar pullorum field isolates and seven reference strains were evaluated. Field isolates were submitted to the National Veterinary Services Laboratories (NVSL), Ames, Iowa, between Oct. 1, 1990, and Aug. 20, 1991. Three antigenic types (standard [n = 96], intermediate [n = 50], and variant [n = 4]) were identified among the field isolates. The NciI fingerprint profiles of all field isolates were identical. Differentiation of field isolates was observed by digestion of DNA with HpaII; three profiles were recognized. One HpaII profile (designated SP HpaII 001) was common among 144 isolates (standard, n = 90; intermediate, n = 50; and variant, n = 4); the remaining six pullorum standard type isolates were equally dispersed between two HpaII profiles (designated SP HpaII 002 and 003). Three antigenic types were identified among the seven reference strains (reference strains 11, 17, 19, and 20 [standard]; reference strain 4 [intermediate]; reference strains 79 and 296 [variant]). One NciI profile was recognized; two HpaII profiles were observed. Six reference strains (11, 17, 19, 20, 79, and 296) had profile SP HpaII 001, and reference strain 4 had profile SP HpaII 002. No correlations were established between DNA fingerprints and antigenic types.