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PubMed2004

DNA fingerprinting and quality traits of Corbarino cherry-like tomato landraces.

Twenty-five landraces of the cherry-like tomato named Corbarino, a typical niche product grown in the Sarno valley of the Campania region, have been characterized. The landraces used have been compared to eight cultivars widely spread in the same area of cultivation. The genetic diversity within and between landraces was evaluated through the comparison of DNA fingerprints obtained with (GATA)(4) probe hybridized to TaqI digested genomic DNA. Twenty-two of the Corbarino landraces were homogeneous and were unequivocally characterized by their DNA fingerprints. The others are probably "population varieties" in that within each of them polymorphic DNA fragments were identified. According to the characterization of fruit shapes, four groups were identified. One landrace from each group, harvested at the same ripening degree, was further characterized for yield, fruit quality components, antioxidant activities, and carotenoid contents and compared to Faino F(1) and Tomito F(1) hybrids. The Corbarino landraces were all characterized by high yield, a high level of carbohydrates, and high levels of soluble and total solids. Interestingly, the biotype named ISCI 05 was the most suitable for canning. Landraces ISCI 07 and ISCI 05 gave the highest value of antioxidant activities and carotenoid content. These data, together with previously published results, suggested that ISCI 05 should be proposed for the institution of a Label of Origin.

DNA Fingerprinting↗
PubMed2001

Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and 16S-23S intergenic spacer and rep-PCR fingerprinting.

The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.

Brazil↗
PubMed1991

Breeding behaviour of pilot whales revealed by DNA fingerprinting.

Most species of whale spend the majority of their lives well away from land, are capable of migrating over large distances and are difficult to identify individually. However, conservation measures require a detailed understanding of their social structure, breeding behaviour and migration patterns. The advent of DNA fingerprinting permits a systematic investigation of such parameters. In the Faeroe Islands there exists a traditional harvest of long-finned pilot whales (Globicephala melas), in which intact social groups (pods) are captured. This affords a unique opportunity to study genetic relationships within and between pods. We report here on a paternity analysis, using DNA fingerprinting, of mother-fetus pairs and males sampled from complete pods. In addition, a single, highly polymorphic minisatellite locus was used to infer degrees of relatedness between groups of fetuses and females. Taken together, our results suggest that pods consist of closely related adult females and their offspring. Sexually mature males either move frequently between pods or remain in their natal pod but refrain from mating with female relatives. Whichever hypothesis is correct, the data suggest that each male spends only a few months with the female post-mating and individual males often father several fetuses within a pod.

Animals↗
PubMed1995

Genetic variation in Phoca vitulina (the harbour seal) revealed by DNA fingerprinting and RAPDs.

Genetic variation in two harbour seal (Phoca vitulina) populations from the Dutch Wadden Sea and Scotland was examined by RAPD analysis and DNA fingerprinting. For comparison a population of grey seals (Halichoerus grypus) was studied. The RAPD method revealed a very low number of polymorphic bands. The multilocus DNA fingerprinting technique showed very low levels of variation both within and between populations of the harbour seals. For one probe a significant difference in variation between populations was demonstrated. The estimated average heterozygosity, however, diverged more strongly between the two harbour seal populations because of more frequent occurrence of rare alleles in the Scottish population. Low levels of genetic variation seem to be characteristic for the subspecies Phoca vitulina vitulina, but not for all marine mammals in the North Sea, as the grey seal population showed significantly higher variation.

Animals↗
PubMed1991

DNA amplification fingerprinting using very short arbitrary oligonucleotide primers.

The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.

Base Sequence↗
PubMed1994

DNA amplification fingerprinting using arbitrary mini-hairpin oligonucleotide primers.

The enzymatic amplification of DNA directed by very short oligonucleotides of arbitrary sequence produces complex DNA profiles useful for genome analysis and identity testing. Mini-hairpins harboring a "core" arbitrary sequence at the 3' terminus primed the amplification of a wide range of templates ranging from plasmid DNA to plant and animal genomes. Primer core regions of only 3 nucleotides produced complex fingerprints, but the hairpin sequence also influenced the amplification reaction. Oligonucleotide substitution with degenerate bases tailored the complexity of fingerprint patterns. Simulation studies of the amplification of plasmids pUC18 and pBR322 using primers with short arbitrary cores allowed assignment of amplification products to expected regions and revealed physical interaction between annealing sites during amplification of first-round products. Mini-hairpin primers can increase detection of polymorphic DNA, and be used to study subgenomic fragments like yeast artificial chromosomes (YACs), cloned DNA and PCR products.

Animals↗
PubMed1992

DNA fingerprinting of oral bacteria.

This elective study was conducted at the Dental School, University of Wales College of Medicine and at the University of Wales College of Cardiff School of Pure and Applied Biology. The student's interest in oral microbiology and desire to contribute to the work being performed in the field of oral microbiology, led him to concentrate his study on developing a DNA fingerprinting method to evaluate the putative new species of the genus Capnocytophaga. The genus Capnocytophaga, which contains Gram-negative, capnophilic fusiform-shaped bacteria, has been implicated in juvenile periodontitis and prepubertal gingivitis. However, the genus is also part of the normal oral flora. The student believed that there was an implication of the existence of both pathogenic and non-pathogenic strains of Capnocytophaga and developed a method of DNA fingerprinting to distinguish Capnocytophaga strains from clusters identified by Khwaja et al. The student felt that the study allowed insight into the tremendous potential of molecular techniques for furthering the understanding of dental disease and confirmed his ambition to pursue a career in academic oral microbiology. This area of study allows new associations to be made between specific bacteria and diseases, aiding the possibility of rapid and early diagnosis.

Blotting, Southern↗
PubMed2002

Phylogeny of bovine species based on AFLP fingerprinting.

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.

Animals↗
PubMed1998

A new fingerprint method for sequence analysis of chromosomal translocations at the genomic DNA level.

Chromosomal rearrangements constitute a significant feature of leukemogenesis and malignant transformation in general. Nucleotide patterns in the immediate vicinity of the break point may provide important information about the underlying causalities, eg illegitimate recombination events mediated by topoisomerase II, Alu repeats, or VDJ recombinase. In order to facilitate the determination of those DNA patterns, we developed a new fingerprint approach. In a first step, two DNA fragments were independently amplified by long distance PCR: the genomic region carrying the break point and the normal nonrearranged counterpart. Subsequently, both PCR products were digested with restriction enzymes, end-labelled with a fluorescent dye, and subjected to high resolution polyacrylamide gel electrophoresis. By comparing the restriction patterns of the rearranged and the nonrearranged PCR fragments, the break points could be easily localized within a size range coverable by a single sequencing reaction. Finally, the exact DNA sequence across the break point was directly determined. The 'fingerprint' technique is fast, reliable and enables the assay of multiple samples in parallel.

Base Sequence↗
PubMed2001

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects.

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability in three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced approximately 50 scoreable polymorphic DNA markers, between individuals of three independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from individual DNA samples that had been combined to create the bulked samples.

Animals↗
PubMed1998

A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites.

Pairwise analysis of HinfI/33.6 DNA fingerprints from a total of one hundred and fifty-three Irish greyhounds of known pedigree were used to determine band-share estimates of unrelated, first-degree and second-degree relationships. Forty-eight unrelated Irish greyhounds were used to determine allele frequencies for three single-locus minisatellites, and following a preliminary screen, eight of the most polymorphic tetra-nucleotide microsatellites from a panel of 15. The results indicated that both band-share estimates by DNA fingerprinting and microsatellite allele frequencies are highly effective in resolving parentage in this greyhound population, while single-locus minisatellites showed limited polymorphism and could not be used alone for routine parentage testing in this breed. The present study also demonstrated that, to obtain optimal resolution of parentage, sample sets of known pedigree status are required to determine the band-share distribution and/or microsatellite allele frequencies.

Animals↗
PubMed2003

DNA fingerprinting of human isolates of Salmonella enterica serotype Paratyphi B in Malaysia.

AIMS: DNA fingerprinting of Salmonella enterica serotype Paratyphi B isolated in Malaysia during 1982-83, 1992 and 1996-2002 was carried out by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility tests and D-tartrate utilization tests to assess the extent of genetic diversity of these isolates in Malaysia. METHODS AND RESULTS: Eighty-six human isolates and one food isolate of Salm. Paratyphi B were analysed by PFGE, antimicrobial susceptibility tests and D-tartrate utilization tests. Sixty-five strains were D-tartrate-negative (dT-) while 22 strains were D-tartrate-positive (dT+). Thirty-seven per cent of the Salm. Paratyphi B strains were resistant to one or more antimicrobial agents. PFGE analysis clearly distinguished the dT- and dT+ strains into two clusters based on the unweighted pair group average method (UPGMA). Twenty-two XbaI-pulsotypes were observed among the 65 dT- strains while 17 XbaI-pulsotypes were observed among the 22 isolates of Salm. Paratyphi B dT+. CONCLUSIONS: The present study showed that PFGE was very discriminative with 33.7% of the strains yielding distinct fingerprints. Paratyphoid fever in Malaysia is probably caused by one predominant, endemic clone of Salm. Paratyphi B dT- with various subtypes. There was no association between the pulsotypes and the severity of the disease indicating that the severity of the disease is probably multifactorial. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing strains of Salm. Paratyphi B. This is the first report on the application of PFGE on a large collection of Salm. Paratyphi B in Malaysia.

Aged↗
PubMed1999

DNA fingerprinting data and the analysis of population genetic structure by comparing band-sharing patterns.

Genetic isolation among populations can be effectively investigated by multilocus DNA fingerprinting. If populations have diverged, it is expected that the mean proportion of bands shared by individuals from the same population, Bw, exceeds the corresponding mean, Bb, calculated from pairs of individuals from distinct populations. A problem arises in deciding whether any difference between Bw and Bb is statistically significant. In fact, any two band-sharing data (bij), contributing to Bw or Bb, are not independent if they share a common individual (like bij and bjl). This prevents a correct application of parametric tests, such as the Student's t-test. Recently, a modification of this test has been proposed that should avoid the independence problem. Using a large number of samples of fingerprints, simulated from an appropriate 'genetic' model, under a wide range of conditions, we compared the performances of the Student's t-test, the modified t-test and five new permutation tests, where individuals, rather than bij values, are permuted. We found that: (i) the Student's t-test can be very permissive, rejecting too often the null hypothesis when true, but is correct or conservative in certain cases; (ii) the modified t-test is extremely conservative when the null hypothesis is true and very inefficient otherwise; (iii) all five permutation tests are strictly correct, provided that individuals are ordered randomly on gels; and (iv) in this case, the permutation tests are equally efficient, and are not inferior to the Student's t-test when the latter is approximately correct and provides a fair benchmark.

DNA Fingerprinting↗
PubMed2003

Sampling strategy in molecular microbial ecology: influence of soil sample size on DNA fingerprinting analysis of fungal and bacterial communities.

Assessing soil microbial community structure by the use of molecular techniques requires a satisfactory sampling strategy that takes into account the high microbial diversity and the heterogeneous distribution of microorganisms in the soil matrix. The influence of the sample size of three different soil types (sand, silt and clay soils) on the DNA yield and analysis of bacterial and fungal community structure were investigated. Six sample sizes from 0.125 g to 4 g were evaluated. The genetic community structure was assessed by automated ribosomal intergenic spacer analysis (A-RISA fingerprint). Variations between bacterial (B-ARISA) and fungal (F-ARISA) community structure were quantified by using principal component analysis (PCA). DNA yields were positively correlated with the sample size for the sandy and silty soils, suggesting an influence of the sample size on DNA recovery, whereas no correlation was observed in the clay soil. B-ARISA was shown to be consistent between the different sample sizes for each soil type indicating that the sampling procedure has no influence on the assessment of bacterial community structure. On the contrary for F-ARISA profiles, strong variations were observed between replicates of the smaller samples (<1 g). Principal component analysis analysis revealed that sampling aliquots of soil > or =1 g are required to obtain robust and reproducible fingerprinting analysis of the genetic structure of fungal communities. However, the smallest samples could be adequate for the detection of minor populations masked by dominant ones in larger samples. The sampling strategy should therefore be different according to the objectives: rather large soil samples (> or =1 g) for a global description of the genetic community structure, or a large number of small soil samples for a more complete inventory of microbial diversity.

Bacteria↗
PubMed2000

Cluster analysis of AP-PCR generated DNA fingerprints of Vibrio vulnificus isolates from patients fatally infected after consumption of raw oysters.

Arbitrarily-primed-polymerase chain reaction (AP-PCR) DNA fingerprints were generated for 10 Vibrio vulnificus strains isolated from patients who became infected and died between 1993 and 1996 as a result of consuming raw oysters. Analysis of the DNA fingerprints with gel imaging and cluster analysis software revealed significant genetic heterogeneity among these strains, suggesting that V. vulnificus has a high degree of variation in its genomic organization, and that multiple pathogenic strains with greatly diverse genomic arrangements, rather than a single type of infective strain or serogroup, caused these infections.

Animals↗
PubMed2001

Allelic deletion fingerprinting of urine cell sediments in bladder cancer.

BACKGROUND: Bladder cancer shows frequent nonrandom allelic deletion at various chromosomal regions. Genotypic detection methods could potentially identify patients at risk for recurrent progressive disease. In this study, we examined allelic deletion at specific chromosomal loci in tumor tissue and urine cell sediment samples using a microsatellite-based protocol. Although both allelic deletion and microsatellite instability have been reported in primary bladder cancer, microsatellite instability was not specifically examined in this study. We report a pilot study of 40 patients with bladder cancer in which allelic deletion in tumor tissue and urine cell sediment was compared with conventional urine cytology results. METHODS AND RESULTS: Forty tumors were analyzed using a set of microsatellite primers from chromosomes 3, 4, 8, 11, 14, and 17 to construct allelic deletion fingerprints. Cy5.5-labeled PCR products were analyzed using the OpenGene System and GeneObjects software. Eighty-eight percent of tumors showed allelic deletion. In urine cell sediments, the tumor detection rate was 80% compared with 50% for routine urine cytology. The allelic deletion fingerprinting (ADF) procedure identified 69% of incipient tumors, cases initially classified as normal by routine urine cytology. CONCLUSION: ADF analysis provides a reliable noninvasive method for the detection and monitoring of recurrent cancer in urine cell sediment samples from patients with bladder cancer.

Adult↗
PubMed2000

Molecular fingerprinting of Mycobacterium tuberculosis strains isolated in Vietnam using IS6110 as probe.

SETTING: Northern and Southern areas of Vietnam. OBJECTIVE: To study the correlation between DNA fingerprinting of 168 Mycobacterium tuberculosis strains isolated from patients with a particular historical past (political separation of Vietnam for 20 years) and data about geographical origin, drug susceptibility, HIV infection and BCG vaccination status. METHODS: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by Southern hybridization of Pvull-digested chromosomal DNA. RESULTS: The number of IS6110 copies for the 168 strains ranges from 0 to 23. Strains originating from the North or the South differ strongly with respect to the number of copies of IS6110. Indeed, the strains originating from the north have predominantly from 3 to 14 IS6110 copies while the southern strains have predominantly from 15 to 23 IS6110 copies. Furthermore, strains isolated in the North are dispersed into 6 groups whereas 80% of the strains isolated in the South form a single group. Moreover, the prevalence of drug resistance is higher in strains isolated in the South than in the North. No noticeable correlation is observed between RFLP patterns, drug susceptibility, or HIV infection. CONCLUSION: The IS6110 fingerprints of 168 M. tuberculosis strains isolated in Vietnam showed a high range of polymorphism. Only a few strains have been found with no IS6110 (1.8%). The differences between the strains from the North and South, having more than six IS6110, suggests that they derived from ancestral strains that would be distinguishable by the number of IS6110 and their transposition sites throughout the genome. The genomic structure of the population of strains from South Vietnam resembles that of the Beijing strain population. This could account for a similar evolution of M. tuberculosis due to a selection by BCG-induced immunity in the two populations.

BCG Vaccine↗
PubMed2005

Molecular relationships in Encephalartos (Zamiaceae, Cycadales) based on nucleotide sequences of nuclear ITS 1&2, rbcL, and genomic ISSR fingerprinting.

The cycad genus Encephalartos is restricted to Africa and is threatened with extinction in most of its range. Total DNA was extracted from 51, i.e., 78 %, of the described species of Encephalartos. The accessions were sampled from the furthest western occurrence of the genus in Nigeria, via Sudan and Uganda, to southern South Africa. The sequences of nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS 1&2), the chloroplast encoded rbcL gene, and ISSR genomic fingerprinting were employed to resolve the molecular history and the relationships within the genus. Sequence alignment, as well as ISSR fingerprinting, data show low genetic variation among all analysed accessions, indicating diversification within the Pliocene/Pleistocene. ITS 1&2 data agree well with morphological and geographical characters and resolved three major genetic clusters with overlapping distribution ranges in eastern South Africa. This area, that contains the largest diversity of genotypes of Encephalartos, may have served as a Pliocene/Pleistocene refugium.

Africa↗