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Identification of sublines of inbred strains of mice and assessment of genetic relationships between substrains or sublines by DNA fingerprinting.

Identification of substrains or sublines of inbred mice and assessment of genetic relationships among them were performed on the basis of DNA fingerprinting using M13 phage DNA as a probe. We used eight C57BL/6 sublines (J parallel Jcl, J parallel Jms, J parallel Slc, J parallel Nrs, J parallel Yok, Jah, N parallel Crj, N parallel Jcl) and eleven C3H/He sublines, (J, J parallel Jcl, J parallel Yok, J parallel Nrs, J parallel Jms, N, N parallel Jcl, N parallel Crj, Slc, Jah, Nrs). Two kinds of restriction endonucleases (HinfI and PstI) were used. It was found that: 1) DNA fingerprint within each subline showed identical patterns. 2) Most sublines of C57BL/6 and C3H/He could be identified using DNA fingerprinting with HinfI except between N parallel Crj and Slc, and among J, J parallel Nrs and J parallel Yok in C3H/He. DNA fingerprints with PstI endonucleases showed low polymorphic banding patterns. 3) A dendrogram constructed from DNA fingerprint patterns reflected generally the genealogy of the sublines used. 4) DNA fingerprinting, therefore, seemed to be suitable for the genetic monitoring and assessment of genetic relationships among sublines of inbred mice having close relationships.

Animals↗

Pseudo-outer product based fuzzy neural network fingerprint verification system.

Fingerprint identification has been used in law enforcement applications over the last century, and has become the de facto international standard for positive identification. With the emergence of automated fingerprint identification technologies, it has assisted in making the once labour-intensive process of classifying, searching and matching a thing of the past. As a biometrics proof of identification, not many have ventured into the world of fingerprint identification using fuzzy neural networks. In this paper, a database of fingerprint images is constructed and a fuzzy neural network called the pseudo outer product fuzzy neural network (POPFNN) [Zhou, R.W. & Quek, C. (1996). A pseudo outer-product based fuzzy neural network. Neural Networks, 9(9), 1569-1581] is trained to detect similarity between two fingerprints and decide whether they belong to the same person. The fundamental idea is that, given a person's fingerprints taken under different conditions, the POPFNN based fingerprint verification system should be sufficiently robust to distinguish the difference. The people providing the fingerprint samples are subjected to different 'adverse' conditions; from wetness to chemical treatments. Fingerprint images are taken after conditions such as: after a shower, holding pineapples (mild acid from fruit), after washing one's hands, etc. The characteristics of POPFNN, such as the learning, generalisation, and high computational abilities, make fingerprint verification particularly powerful when verifying authentic fingerprints subjected to external conditions and recognising spurious ones. In order to demonstrate the efficacy of POPFNN and its application in the fingerprint verification system (FVS), several types of experiments have been designed and implemented in this work. The experimental results and analysis are presented at the end of the paper for discussion.

Algorithms↗

Precalibration of matrix-assisted laser desorption/ionization-time of flight spectra for peptide mass fingerprinting.

Identification of proteins using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide mass fingerprinting (PMF) is a key technique in proteomics. The method is known to be sensitive as well as amenable to high-throughput operation, but the resulting identifications suffer from a relatively low level of confidence. One way of increasing the confidence is by improving measurement accuracy using one of several calibration methods. This paper presents a new strategy for calibration of MALDI-TOF PMF spectra that makes use of the phenomenon of peptide mass clustering, and enables spectrum calibration prior to the step of database interrogation, before or after peak extraction. Typically, mass errors are reduced by 40-60%. Accuracy improvement at this early stage can help avoid losing protein candidates, reduce the number of external calibration spots, eliminate internal calibrants, and reduce the number of candidates being scored, thereby reducing analysis time. Different variants of the method are discussed and compared to known calibration methods, such as relying on known calibrants or comparison to putative database candidates. In order to allow precise description of the method and to place the results in perspective, theoretical considerations of peptide databases and scoring functions are also discussed.

Algorithms↗

Improving automatic peptide mass fingerprint protein identification by combining many peak sets.

An automated peak picking strategy is presented where several peak sets with different signal-to-noise levels are combined to form a more reliable statement on the protein identity. The strategy is compared against both manual peak picking and industry standard automated peak picking on a set of mass spectra obtained after tryptic in gel digestion of 2D-gel samples from human fetal fibroblasts. The set of spectra contain samples ranging from strong to weak spectra, and the proposed multiple-scale method is shown to be much better on weak spectra than the industry standard method and a human operator, and equal in performance to these on strong and medium strong spectra. It is also demonstrated that peak sets selected by a human operator display a considerable variability and that it is impossible to speak of a single "true" peak set for a given spectrum. The described multiple-scale strategy both avoids time-consuming parameter tuning and exceeds the human operator in protein identification efficiency. The strategy therefore promises reliable automated user-independent protein identification using peptide mass fingerprints.

Cell Line↗

Generation of DNA probes for detection of microorganisms by polymerase chain reaction fingerprinting.

Identification of medically relevant microorganisms is important for diagnosis, treatment and prevention of infectious diseases. This has initiated the development of a large number of identification and typing techniques based on phenotypic and genetic characteristics. In general, these last mentioned nucleic acid-mediated techniques provide more detailed and consistent information on strain-specific characteristics. However, the development of clinically useful microbial DNA/RNA probes requires nucleotide sequence information and a set of well defined reference organisms for test validation in comparison with the current gold standard. This is a requirement for the development of accurate nucleic acid hybridisation and/or amplification tests. Recently, it has been demonstrated that polymerase chain reaction (PCR)-mediated genetic typing of microorganisms can lead to the immediate isolation of species-specific DNA probes by comparison of DNA fingerprints. This combines the sensitivity of PCR with the specificity of DNA probing without the need to generate nucleic acid sequence information prior to probe development. The implications of this procedure for clinical microbiology and epidemiological surveillance will be discussed. It is shown that specific probes can be developed for various taxonomic levels and that detection and identification can be combined into a single, fast procedure. The versatility and widely applicable principles of this procedure will be highlighted and exemplified by some newly developed tests and a review of the current literature.

Bacteria↗

Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification.

Fingerprints for 72 reference serovar strains of pathogenic Leptospira spp. were obtained by pulsed-field gel electrophoresis (PFGE) following NotI restriction digests of the chromosome. These strains included the serovar reference strains of serogroups Australis, Ballum, Bataviae, Grippotyphosa, Panama, Pomona, and Pyrogenes. Sixty-four serovars could be identified by a unique NotI restriction profile. The remaining serovars were differentiated by chromosomal digestion with SgrAI. These included four serovars from serogroup Australis, two serovars from serogroup Ballum, and two serovars from serogroup Bataviae. Thirteen of 18 recent clinical isolates identified by microagglutination test and cross-adsorption procedure were correctly typed by PFGE. The results indicate that PFGE, which is considerably more rapid than serology, should be useful for identification and epidemiological studies.

Animals↗

The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce group-specific fingerprints.

Identification of microbial community members in complex environmental samples is time consuming and repetitive. Here, ribosomal sequences and hidden Markov models are used in a novel approach to rapidly assign fungi to their presumptive genera. The ITS1 and ITS2 fragments from a range of axenic, anaerobic gut fungal cultures, including several type strains, were isolated and the RNA secondary structures predicted for these sequences were used to generate a fingerprinting program. The methodology was then tested and the algorithms improved using a collection of environmentally derived sequences, providing a rapid indicator of the fungal diversity and numbers of novel sequence groups within the environmental sample from which they were derived. While the methodology was developed to assist in investigations involving the rumen ecosystem, it has potential generic application in studying diversity and population dynamics in other microbial ecosystems.

Animals↗

Development of oil hydrocarbon fingerprinting and identification techniques.

Oil, refined product, and pyrogenic hydrocarbons are the most frequently discovered contaminants in the environment. To effectively determine the fate of spilled oil in the environment and to successfully identify source(s) of spilled oil and petroleum products is, therefore, extremely important in many oil-related environmental studies and liability cases. This article briefly reviews the recent development of chemical analysis methodologies which are most frequently used in oil spill characterization and identification studies and environmental forensic investigations. The fingerprinting and data interpretation techniques discussed include oil spill identification protocol, tiered analytical approach, generic features and chemical composition of oils, effects of weathering on hydrocarbon fingerprinting, recognition of distribution patterns of petroleum hydrocarbons, oil type screening and differentiation, analysis of "source-specific marker" compounds, determination of diagnostic ratios of specific oil constituents, stable isotopic analysis, application of various statistical and numerical analysis tools, and application of other analytical techniques. The issue of how biogenic and pyrogenic hydrocarbons are distinguished from petrogenic hydrocarbons is also addressed.

Accidents↗

Peptide mass fingerprinting: protein identification using MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-staining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.

Databases, Protein↗

Pulsed-field gel electrophoresis fingerprinting for identification of Azospirillum species.

Pulsed-field gel electrophoresis (PFGE) was used to obtain macrorestriction fingerprints of restriction enzyme-cut DNA of natural isolates of Azospirillum spp. Metabolic profiles, along with other phenotypic characteristics, were compared with these fingerprints to differentiate among the azospirilla isolates. A wide diversity of phenotypes (e.g., colony color, motility, and accumulation of poly-beta-hydroxybutyrate granules) was observed among the natural isolates of azospirilla. PFGE revealed that TCTAGA, the sequence recognized by Xba1, is rare in the genome of azospirilla. The PFGE fingerprint revealed that azospirilla associated with different crops have a very similar genetic background. PFGE fingerprints were more consistent in the identification of azospirilla isolates from specific hosts than the metabolic fingerprints. For further differentiation at strain level, metabolic, physiological, and morphological profiles provide additional information.

Azospirillum↗

Applicability of rep-PCR fingerprinting for identification of Lactobacillus species.

PCR amplification of repetitive bacterial DNA elements fingerprinting using the (GTG)(5) primer ((GTG)(5)-PCR) was proven to be useful for differentiation of a wide range of lactobacilli (i.e. 26 different (sub)species) at the species, subspecies and potentially up to the strain level. Using this rapid and reproducible genotypic technique, new Lactobacillus isolates recovered from different types of fermented dry sausage could be reliable identified at the (sub)species level. In conclusion, (GTG)(5)-PCR was found to be a promising genotypic tool for rapid and reliable speciation and typing of lactobacilli and other lactic acid bacteria important in food-fermentation industries.

Animals↗

Rapid DNA mutation identification and fingerprinting using base excision sequence scanning.

Base excision sequence scanning (BESS) is a new polymerase chain reaction (PCR)-based mutation scanning method that locates and identifies all DNA mutations. The BESS method consists of two procedures that generate "T" (BESS T-Scan) and "G" ladders (BESS G-Tracker) analogous to T and G ladders of dideoxy sequencing. The BESS procedures are simple to perform and require no special equipment or gels, no reaction optimization beyond PCR, and no heteroduplex formation. The samples are analyzed on standard sequencing gels or on automated DNA sequencers, and the data produced are easy to interpret, requiring a simple comparison of the sequence of normal and mutant DNA. The BESS method is versatile, having applications not only for mutation detection, but also single nucleotide polymorphism (SNP) discovery and analysis, DNA fingerprinting (including viral and bacterial typing), and clone identification. In this study, we utilize BESS in two of these applications: detection of a point mutation in BRCA1, and DNA typing of human papilloma virus (HPV).

BRCA1 Protein↗

FindPept, a tool to identify unmatched masses in peptide mass fingerprinting protein identification.

FindPept (http://www.expasy.org/tools/findpept.html) is a software tool designed to identify the origin of peptide masses obtained by peptide mass fingerprinting which are not matched by existing protein identification tools. It identifies masses resulting from unspecific proteolytic cleavage, missed cleavage, protease autolysis or keratin contaminants. It also takes into account post-translational modifications derived from the annotation of the SWISS-PROT database or supplied by the user, and chemical modifications of peptides. Based on a number of experimental examples, we show that the commonly held rules for the specificity of tryptic cleavage are an oversimplification, mainly because of effects of neighboring residues, experimental conditions, and contaminants present in the enzyme sample.

Algorithms↗

The development of a matrix-assisted laser desorption/ionization mass spectrometry-based method for the protein fingerprinting and identification of Aeromonas species using whole cells.

This report describes the development of a method to detect the waterborne pathogen Aeromonas using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, MALDI-MS was applied to the characterization of seventeen species of Aeromonas. These seventeen species were represented by thirty-two strains, which included type, reference and clinical isolates. Intact cells from each strain were used to generate a reproducible library of protein mass spectral fingerprints or m/z signatures. Under the test conditions used, peak lists of the mass ions observed in each species revealed that three mass ions were conserved among all the seventeen species tested. These common mass ions having an average m/z of 6301, 12,160 or 12,254, and 13,450, can be potentially used as genus-specific biomarkers to identify Aeromonas in unknown samples. A dendrogram generated using the m/z signatures of all the strains tested indicated that the mass spectral data contained sufficient information to distinguish between genera, species, and strains. There are several advantages of using MALDI-MS based protein mass spectral fingerprinting of whole cells for the identification of microorganisms as well as for their differentiation at the sub-species level: (1) the capability to detect proteins, (2) high throughput, and (3) relatively simple sample preparation techniques. The accuracy and speed with which data can be obtained makes MALDI-MS a powerful tool especially suited for environmental monitoring and detection of biological hazards.

Aeromonas↗

Identification and DNA fingerprinting of Legionella strains by randomly amplified polymorphic DNA analysis.

The randomly amplified polymorphic DNA (RAPD) technique was used in the development of a fingerprinting (typing) and identification protocol for Legionella strains. Twenty decamer random oligonucleotide primers were screened for their discriminatory abilities. Two candidate primers were selected. By using a combination of these primers, RAPD analysis allowed for the differentiation between all different species, between the serogroups, and further differentiation between subtypes of the same serogroup. The usefulness of RAPD analysis was also evaluated with outbreak-related clinical and environmental isolates previously typed by the restriction fragment length polymorphism technique. RAPD analysis proved to be as accurate as other genotypic methods, reproducible, and highly discriminatory and is a valuable new alternative to traditional fingerprinting and identification of Legionella species and strains.

Animals↗

Comparison of amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism fingerprinting for identification of Acinetobacter genomic species and typing of Acinetobacter baumannii.

Thirty-one strains of Acinetobacter species, including type strains of the 18 genomic species and 13 clinical isolates, were compared by amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA analysis (RAPD), and amplified fragment length polymorphism (AFLP) fingerprinting. ARDRA, performed with five different enzymes, showed low discriminatory power for differentiating Acinetobacter at the species and strain level. The standardized commercially available RAPD kit clearly enabled the discrimination of all Acinetobacter genomic species but showed great polymorphism between isolates of Acinetobacter baumannii. AFLP fingerprinting with radioactively as well as fluorescently labelled primers showed high discriminatory power for the identification of 18 Acinetobacter genomic species and typing of 13 clinical Acinetobacter isolates. Compared to radioactive AFLP, fluorescent AFLP was technically fast and simple to perform, and it permitted analysis with an automated DNA sequencer. Fluorescent AFLP seems particularly well suited for studying the epidemiology of nosocomial infections and outbreaks caused by Acinetobacter species.

Acinetobacter↗