Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) for typing strains of vancomycin-resistant Enterococcus faecium

In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of a novel assay based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) was tested. A large number of vancomycin-resistant Enterococcus faecium (VREM) isolates from haematological ward patients of the Clinical Hospital in Gdańsk were examined. We found that ADSRRS fingerprinting analysis is a rapid method that offers good discriminatory power. The method demonstrated also excellent reproducibility. The usefulness of the ADSRRS fingerprinting method for molecular typing was compared with pulsed field gel electrophoresis (PFGE) method, which is currently considered the gold standard for molecular typing of isolates recovered from patients and the environment in the course of investigation and control of nosocomial outbreaks. Clustering of ADSRRS fingerprinting data matched pulsed field gel electrophoresis data.The features of ADSRRS fingerprinting technique is discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning VREM that may occur in a single medical ward.     

Assessment of discriminatory power of three different fingerprinting methods based on killer toxin sensitivity for the differentiation of Saccharomyces cerevisiae strains

AIMS: A panel composed of 44 taxonomically certified strains of Saccharomyces cerevisiae of different origin was used to evaluate the discriminatory power of three different fingerprinting methods based on sensitivity towards 24 killer toxins. METHODS AND RESULTS: Binary data matrix (BDM), triplet data matrix (TDM) and numerical data matrix (NDM) were used as fingerprinting methods. NDM possessed the highest discriminatory power, assessed through the Simpson's, and Hunter and Gaston's indices for the measurement of diversity. The upper limits of fingerprinting ability expressed by the three above methods have been also discussed. CONCLUSIONS: NDM determined a significant increase of discriminatory power than the use of BDM or TDM, in terms of an effective amplification of their fingerprinting efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The NDM fingerprinting method could find application in control laboratories for the discrimination of yeast strains of industrial importance or covered by patent.     

The evolving technology of DNA fingerprinting and its application to fisheries and aquaculture

In 1985, Alec Jeffreys reported the development of multilocus DNA fingerprinting by Southern blot-detection of hypervariable minisatellites or variable number of tandem repeat (VNTR) loci. This technology found immediate application to various forensic and scientific problems, including fisheries and aquaculture. By 1989, however, it was recognized by many researchers that inherent problems exist in the application of multilocus fingerprinting to large sample sizes as might occur in fisheries and aquaculture genetic studies. As such, individual VNTRs were cloned for single-locus DNA fingerprinting. Although single-locus fingerprinting ameliorates many of the problems associated with multilocus DNA fingerprinting, it suffers from the problem that electrophorectic anomalies of band migration within and between gels necessitates binning of alleles, thus underestimating genetic variability in a given population. Amplification of microsatellite loci by the polymerase chain reaction, however, solved many of the problems of Southern blot-based DNA fingerprinting. Moreover, microsatellites exhibit attributes that make them particularly suitable as genetic markers for numerous applications in aquaculture and fisheries research: (1) they are abundant in the genome; (2) they display varying levels of polymorphism; (3) alleles exhibit codominant Mendelian inheritance; (4) minute amounts of tissue are required for assay (e.g., dried scales or otoliths); (5) loci are conserved in related species; (6) potential for automated assay. Recent innovations in DNA fingerprinting technology developed over the past 5 years are discussed with special emphasis on microsatellites and their application to fisheries and aquaculture, e.g., behavioural and population genetics of wild species, and selection and breeding programmes for aquaculture broodstock.     

Metabolic fingerprinting of blood plasma from patients with prostate cancer

Application of blood plasma metabolites fingerprinting for the diagnostic of the 2nd stage of prostate cancer has been investigated. The diagnostic sensitivity (95%), specificity (96.7%) and accuracy (95.7%) of the metabolic fingerprinting were much higher then those for the ELISA PSA test (35%, 83.3% and 51.7%, respectively) performed for the same patients. Area under the ROC curve (0.994) suggests that the proposed approach based on the metabolic fingerprinting is effective and applicable in clinical practice.     

Comparison of molecular fingerprinting methods for analysis of soil microbial community structure

Denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) analysis, and automated ribosomal intergenic spacer analysis (ARISA) have been widely used as molecular fingerprinting methods for analysis of microbial communities. To find suitable methods, we compared the three fingerprinting methods by analyzing soil fungal communities in four differing land-use types: bare ground, crop fields, grasslands, and forests. We also examined optimal primer pairs for DGGE analysis by comparing single and mixed DNA samples of cultured fungal populations. Principal coordinate analysis (PCO), nonmetric multidimensional scaling method (NMDS), and analysis of similarities (ANOSIM), which are major multivariate statistical analyses for quantifying fingerprint patterns, were compared. All three fingerprinting methods yielded clear discrimination of soil fungal communities among the four land-use types, irrespective of statistical methods. The advantages and disadvantages of the three fingerprinting methods were discussed.     

Orthogonal array design in optimizing ERIC-PCR system for fingerprinting rat's intestinal microflora

AIMS: The aim of the present study was to rapidly optimize enterobacterial repetitive intergenic consensus (ERIC)-PCR amplification systems for fingerprinting rat's intestinal microflora. METHODS AND RESULTS: Orthogonal array design and statistic analysis methods were attempted to rapidly optimize ERIC-PCR reaction system for fingerprinting intestinal microflora. The results showed that variations of the four factors (Mg(2+), dNTP, primer and HotstarTaq polymerase concentrations) changed the fingerprinting patterns significantly. The order of effects of those factors on fingerprinting patterns was primers (F = 274.000, P = 0.000), Hotstar Taq polymerase (F = 197.000, P = 0.001), Mg(2+) (F = 181.000, P = 0.001) and dNTP (F = 27.000, P = 0.011). The optimal ERIC-PCR condition was containing 200 micromol l(-1) dNTP, 2.5 mmol l(-1) Mg(2+), 0.4 micromol l(-1) primer, 1 U HotstarTaq DNA polymerase namely 25 microl reaction system, which is proved to be a simple, fast and reliable method suitable for fingerprinting rat's intestinal microflora. CONCLUSIONS: The results suggest that Mg(2+), dNTP, primer and HotstarTaq polymerase concentrations play important roles on ERIC-PCR fingerprinting patterns. Orthogonal array design is a considerable method to optimize ERIC-PCR reaction system for its rapidness, simplicity, potential to investigate mutual effects of parameters. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first report on optimization of ERIC-PCR amplification systems for fingerprinting intestinal microflora using orthogonal array design or statistic analysis methods and systematically observing the effects of variables of reaction conditions.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

AFLP在分子生物学研究中的应用

扩增片段长度多态性（AFLP）可靠性强,多态检出率高,因而被认为是最有效的DNA指纹分析技术。AFLP已广泛应用于分类学、病理学、种群遗传学、DNA指纹分析的研究和建立数量性状基因图谱,成为最主要的遗传标记。介绍了AFLP的原理、影响因素及其在分子生物学研究中的应用。     

High-resolution genotyping of Salmonella strains by AFLP-fingerprinting

High resolution AFLP fingerprinting, in which subsets of genomic restriction fragments are amplified by means of PCR, was used for the identification of different Salmonella serotypes to investigate whether this technique is applicable in epidemiological studies. Seventy-eight different Salmonella strains comprising 62 serotypes were genetically identified by AFLP. Primer combination M00 ( Mse I primer without additional 3' nucleotides) and E11 ( Eco RI primer with two additional 3' nucleotides) resulted in reproducible profiles containing approximately 50 bands. All serotypes were characterized by a unique profile. In addition, AFLP fingerprinting enabled phage type identification. Different strains previously identified as identical, using typing methods with lower resolution, could be distinguished, showing that AFLP fingerprinting is well suited for bacterial epidemiology and identification.     

SRFA法构建水稻DNA指纹图谱

水稻基因组DNA用PstⅠ酶切同时与人工接头连接后,使用选择性引物进行PCR扩增,琼脂糖凝胶电泳检测所构建的水稻DNA指纹图谱。结果表明在JX17和ZYQ8间以及5种野生稻间均存在DNA多态性片段。     

DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

Summary Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms. Fourteen human cell lines previously found by other methods to be either related or disparate were subjected to DNA fingerprinting by PCR amplification of selected fragment length polymorphism loci. Cell identification patterns by this method were concordant with those obtained by isoenzyme phenotyping and restriction fragment length polymorphism-DNA fingerprinting, and were reproducible within and between assays on different DNA extracts of the same cell line. High precision was achieved with electrophoretic separation of amplified DNA products on high resolution agarose or polyacrylamide gels, and with fragment length polymorphism (FLP) loci-specific “allelic ladders” to identify individual FLP alleles. Determination of the composite fingerprint of a cell line at six appropriately chosen fragment length polymorphism loci should achieve a minimum discrimination power of 0.999. The ability of PCR-based fragment length polymorphism DNA fingerprinting to precisely and accurately identify the alleles of different human cell lines at multiple polymorphic fragment length polymorphism loci demonstrates the feasibility of developing a cell line DNA fingerprint reference database as a powerful additional tool for future cell line identification and authentication.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

An empirical study of web browsers’ resistance to traffic analysis and website fingerprinting attacks

Anonymity protocols are employed to establish encrypted tunnels to protect the privacy of Internet users from traffic analysis attacks. However, the attackers strive to infer some traffic patterns’ characteristics (e.g. packet directions, packet sizes, inter-packet timing, etc.) in order to expose the identities of Internet users and their activities. A recent and popular traffic analysis attack is called website fingerprinting which reveals the identity of websites visited by target users. Existing work in the literature studied the website fingerprinting attack using a single web browser, namely Firefox. In this paper we propose a unified traffic analysis attack model composed of a sequence of phases that demonstrate the efficiency of website fingerprinting attack using popular web browsers under Tor (The Onion Router). In addition, we reveal the main factors that affect the accuracy of website fingerprinting attack over Tor anonymous system and using different browsers. To the best of our knowledge, no previous study uncovered such factors by deploying real-world traffic analysis attack utilizing the top five web browsers. The outcomes of the research are very relevant to Internet users (individuals/companies/governments) since they allow to assess to which extent their privacy is preserved in presence of traffic analysis attacks, in particular, website fingerprinting over different browsers. A recommendation for future research direction regarding the investigation of website fingerprinting over different scenarios is also provided.     

Evaluation of (GTG)<Subscript>5</Subscript>-PCR for rapid identification of <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using the (GTG)(5) primer was applied for fast screening of bacterial strains isolated from dental plaque of early childhood caries (ECC)-affected children. A group of 29 Gram-positive bacteria was separated into a homogeneous cluster together with Streptococcus mutans reference strains and constituted an aberrant branch after the numerical analysis of (GTG)(5)-PCR fingerprints. Automated ribotyping with EcoRI restriction enzyme (RiboPrinter microbial characterization system) revealed high genetic heterogeneity among the tested group and proved to be a good tool for strain-typing purposes. Further characterization of the studied strains was achieved by extensive phenotyping and whole-cell protein fingerprinting and confirmed all the strains as S. mutans representatives. Obtained results showed rep-PCR fingerprinting with the (GTG)(5) primer to be a fast and reliable method for identification of S. mutans.     

Typing of Saccharomyces cerevisiae and Kloeckera apiculata strains from Aglianico wine

AIMS: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. METHODS AND RESULTS: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. CONCLUSIONS: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.     

Comparative assessment of ISSR, DAMD and SCoT markers for evaluation of genetic diversity and conservation of landrace chickpea (Cicer arietinum L.) genotypes collected from north-west of Iran

Morphological traits and three molecular markers techniques: start codon targeted (SCoT), inter-simple sequence repeat (ISSR) and directly amplified minisatellite DNA (DAMD) markers were compared for fingerprinting of 40 landraces chickpea genotypes collected from different geographical locations of north-west of Iran. Variance analysis of ten measured morphological traits showed significant differences existed between genotypes. Cluster analysis based on morphological traits, divided genotypes in three distinct clusters. Average polymorphism information content (PIC) for ISSR, DAMD and SCoT markers was 0.216, 0.232 and 0.232, respectively, and this revealed that SCoT markers were more informative, followed by ISSRs marker, than other markers for the assessment of diversity amongst genotypes. Cluster analysis for three different molecular types revealed that genotypes taken for the analysis can be divided in three and four distinct clusters. Accessions from same geographical regions mostly showed more genetic similarities than those from origins far isolated apart. These results suggest that efficiency of SCOT, DAMD and ISSR markers was relatively the same in fingerprinting of genotypes but SCOT and DAMD analysis are more effective in fingerprinting of chickpea genotypes. To our knowledge, this is the first detailed report of a comparison of performance among two targeted DNA region molecular markers (SCoT and DAMD) and the ISSR technique on a set of samples of chickpea. Overall, our results indicate that SCOT, ISSR and DAMD fingerprinting could be used to detect polymorphism for genotypes of chickpea.     

Application of a clustering-based peak alignment algorithm to analyze various DNA fingerprinting data

DNA fingerprinting analysis such as amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic PCR (rep-PCR), ribosomal intergenic spacer analysis (RISA), and denaturing gradient gel electrophoresis (DGGE) are frequently used in various fields of microbiology. The major difficulty in DNA fingerprinting data analysis is the alignment of multiple peak sets. We report here an R program for a clustering-based peak alignment algorithm, and its application to analyze various DNA fingerprinting data, such as ARDRA, rep-PCR, RISA, and DGGE data. The results obtained by our clustering algorithm and by BioNumerics software showed high similarity. Since several R packages have been established to statistically analyze various biological data, the distance matrix obtained by our R program can be used for subsequent statistical analyses, some of which were not previously performed but are useful in DNA fingerprinting studies.     

Sequence analysis of T1 ribonuclease fragments of 18S ribosomal RNA by 5''-terminal labeling, partial digestion, and homochromatography fingerprinting.

The method employed to determine the sequence of a T1 RNase fragment, A-A-A-A-A-U-A-A-C-A-A-U-A-C-A-Gp, from Novikoff rat hepatoma 18S ribosomal RNA is described. This method is applicable to any oligoribonucleotide produced by specific endonucleases that leave the newly cleaved 5'-end free for labeling with polynucleotide kinase and gamma-(32p)-ATP. The (32p)-labeled oligoribonucleotide is subjected to partial endonucleolytic digestion and fractionated by two-dimensional homochromatography fingerprinting. The nucleotide sequence is determined by following mobility shifts of the labeled and partially digested oligoribonucleotides in homochromatography fingerprinting.