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At least 127 records · Page 7Linked to original sources
PubMed1989

Protein fingerprinting: a novel virus identification system.

Viral proteins separated by one-dimensional SDS-PAGE produce protein binding patterns (fingerprints) which are unique for different viruses. We have applied this concept successfully for the development of a practical and objective virus identification system which is applicable to most viruses. The method is simple, specific, and, unlike the currently available methods, free from all virus-specific reagents. Interference by host protein bands in SDS-PAGE preparations of virus-infected cell lysates was eliminated consistently by treating virus infected cell cultures with optimum concentration of NaCl for selective inhibition of host protein synthesis. The method utilizes the comparison of protein fingerprints of 'unknown' viruses with protein fingerprints of reference viruses stored in a computer data base, using pattern recognition software. All 113 'unknown' virus strains were correctly identified to the genus level by the protein fingerprint method, when compared with the conventional virus identification methods.

Animals↗
PubMed2002

Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting.

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.

Amino Acid Sequence↗
PubMed2003

Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-alpha-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging.

Amino Acid Sequence↗
PubMed2001

A novel toxicity fingerprinting method for pollutant identification with lux-marked biosensors.

A novel technique is described for the identification and quantification of environmental pollutants based on toxicity fingerprinting with a metabolic lux-marked bacterial biosensor. This method involved characterizing the toxicity-based responses of the biosensor to seven calibration pollutants as acute temporal-dose response fingerprints. An algorithm is described to allow comparisons of responses of an unknown pollutant to be made against the calibration data. This is based on predicting pollutant concentration at each of six different time points over the course of a 5-min assay. If the prediction is consistent between the unknown pollutant and a calibration pollutant at the 95% test level, this is considered to be a positive identification. All seven calibration pollutants could be successfully distinguished from each other with this technique. Environmental samples, individually spiked with single concentrations of pollutants, were compared in this way against the calibration pollutants. An 83% identification success was achieved, with no false positives at the 95% test level. This is a simple and rapid technique that potentially can be applied to monitoring of industrial wastewater or as a screening tool for regulators.

Algorithms↗
PubMed1999

[Methodology of DNA fingerprinting and its application in identification of M. tuberculosis].

OBJECTIVE: To explore methodology of DNA fingerprinting technique and its application in identification of strains of M. tuberculosis. METHODS: This research was based on different copy numbers and location of chromosome DNA IS6110 in M. tuberculosis genome. After DNA of M. tuberculosis were cut by endonuclease PvuII, the products were transferred to nylon membrane, then detected and hybridized by enhanced chemiluminescent (ECL) labeling techniques. RESULTS: Fifty clinical isolates from different TB patients had unidentical DNA fingerprinting patterns. DNA fingerprinting patterns showed a great difference from sputum and pus specimens of the same patient. H(37) R(v) ofloxacin resistant and sensitive strains had an identical DNA fingerprinting pattern. CONCLUSIONS: Identification of M. tuberculosis strains is feasible by DNA fingerprinting technique.

DNA Fingerprinting↗
PubMed1997

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting.

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Chromosomes, Artificial, Yeast↗
PubMed2004

Taxonomic relationship among seven species of groupers (genus Epinephelus; family Serranidae) as revealed by RAPD fingerprinting.

Phenotypic identification of groupers of the genus Epinephelus is based on color patterns and a suite of morphologic characters. However, these characters often show intraspecific variations and differences between juveniles and adults of the same species. The present work is an attempt to study and ratify the status of Epinephelus spp. using random amplified polymorphic DNA (RAPD) analysis based on samples drawn from southeast and southwest coasts of India. The RAPD fingerprints generated in Epinephelus diacanthus, E. areolatus, E. chlorostigma, E. bleekeri, E. coioides, E. tauvina, and E. malabaricus with 4 primers (OPA 01, OPA 07, OPF 08, and OPF 10) were consistent, reproducible, and yielded species-specific diagnostic markers in all the species. A total of 59 RAPD loci in the size range of 70 to 4500 bp were produced from all 4 arbitrary primers. An unweighted pair-group method with arithmetic mean (UPGMA) dendogram was constructed based on genetic distance values to show the genetic relationships among the 7 species. All the individuals of each species formed monophyletic species clusters. The mean intraspecies genetic distance value (0.305) was significantly lower than the interspecies value (0.365). Epinephelus malabaricus was most distantly related to E. diacanthus and E. bleekeri. The genetic relationship was very close among E. coioides, E. tauvina, and E. malabaricus and also between E. chlorostigma and E. bleekeri. Within-species genetic polymorphism was highest in E. chlorostigma (49.15%) and lowest in E. tauvina (25.42%).

Animals↗
PubMed2005

Effects of varied pH, growth rate and temperature using controlled fermentation and batch culture on matrix assisted laser desorption/ionization whole cell protein fingerprints.

Rapid identification of microorganisms using matrix assisted laser desorption/ionization (MALDI) is a rapidly growing area of research due to the minimal sample preparation, speed of analysis and broad applicability of the technique. This approach relies on expressed biochemical markers, often proteins, to identify microorganisms. Therefore, variations in culture conditions that affect protein expression may limit the ability of MALDI-MS to correctly identify an organism. We have expanded our efforts to investigate the effects of culture conditions on MALDI-MS signatures to specifically examine the effects of pH, growth rate and temperature. Continuous cultures maintained in bioreactors were used to maintain specific growth rates and pH for E. coli HB 101. Despite measurable morphological differences between growth conditions, the MALDI-MS data associated each culture with the appropriate library entry (E. coli HB 101 generated using batch culture on a LB media), independent of pH or growth rate. The lone exception was for a biofilm sample collected from one of the reactors which had no appreciable degree of association with the correct library entry. Within the data set for planktonic organisms, variations in growth rate created the largest variation between fingerprints. The effect of varying growth temperature on Y. enterocolitica was also examined. While the anticipated effects on phenotype were observed, the MALDI-MS technique provided the proper identification.

Bacterial Proteins↗
PubMed1993

RAPD fingerprinting is useful for identification of Azospirillum strains.

In vitro amplification of genomic DNA fragments with single primers of arbitrary sequence was used as a rapid and sensitive method to obtain fingerprints of ten strains belonging to three of the Azospirillum species: brasilense, lipoferum and amazonense. Each strain showed a distinctive pattern of bands that permitted its unequivocal identification. Closely related strains produced almost identical fingerprints. Pairwise comparison and cluster analysis of the amplification patterns allowed grouping of the strains. The resulting dendrograms are similar to previous dendrograms based on the restriction endonuclease analysis (REA) of total DNA and on the restriction fragment length polymorphism (RFLP). Our results indicate that the random amplified polymorphic DNA (RAPD) technique is a simple, fast and useful tool for the determination of genetic relationships among Azospirillum isolates and to evaluate the genomic stability of the Azospirillum strains released in the environment.

Azospirillum↗
PubMed1998

Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates.

Four PCR-based DNA fingerprinting techniques were compared for their ability to identify at the species level a heterogeneous collection of isolates belonging to the six valid Listeria species. 16S rDNA-RFLP analysis identified all species and 16S rDNA-SSCP analysis identified almost all species. Also, isolates with unusual biochemical characteristics and/or unusual antigenic composition could be identified correctly. rRNA-intracistronic length polymorphism analysis suffered from high intraspecific variability, a limited number of fragments per profile, and small length differences between the spacers of different species. tRNA-intergenic length polymorphism analysis resulted in identification of all isolates but one, when fluorescent DNA capillary electrophoresis was used such that fragment length differences of 1 bp could be resolved. The four techniques yielded comparable results relevant to the taxonomy of Listeria. They all indicate a high degree of genetic relatedness between L. innocua and L. welshimeri, homogeneity of L. grayi, distinct but clear relatedness of L. grayi to the other Listeria species, a clear distinction between the two subspecies of L. ivanovii, and a clear distinction between Listeria isolates and isolates from closely related taxa or from species which are phenotypically difficult to distinguish from Listeria. New sequence determination of the 16S rRNA gene was necessary to obtain sequences in accordance with the findings of 16S rDNA-RFLP analysis.

DNA Fingerprinting↗
PubMed1994

Random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) fingerprints in forensic species identification.

The random amplified polymorphic DNA (RAPD) method was used to identify the species of forensic biological samples. Neither genomic DNA sequence nor two polymerase chain reaction (PCR) cycle programs is required. Only single 10-nt primer and one PCR program are used. We demonstrated that this method can be used to identify animals including bovine, goat, pig, dog, rat, rabbit, chicken, duck, and human by comparing their RAPD PCR fingerprints. This study provides a simple, fast and sensitive fingerprinting method in species identification for crime scene evidence or food products of endangered species.

Animals↗
PubMed1989

Individual identification from semen by the deoxyribonucleic acid (DNA) fingerprint technique.

For individual identification from semen, the deoxyribonucleic acid (DNA) fingerprint technique was used. In a blind trial, we succeeded in determining the semen donors among several volunteers comparing the DNA fingerprints of the blood and semen samples, respectively. Thereafter, we examined semen in a condom left beside a naked female dead body. The DNA fingerprint of the semen was recognized to be identical to that of the blood from a suspected man arrested later. This is the first report that the DNA fingerprint technique was practically used in a criminal investigation in Japan.

DNA↗
PubMed1992

DNA fingerprints: a tool for identification and determination of the relationships between species and strains of Leishmania.

Using nonradioactive hybridization, the multilocal probes 33.15, F10 and (CAC)5 were shown to recognize multiple minisatellite regions in nuclear DNA of Leishmania, producing on Southern blots complex banding patterns typical of DNA fingerprints. We used the 33.15 probe to study 14 different strains belonging to 6 different species from both the sub-genus Viannia and the Leishmania mexicana complex of the sub-genus Leishmania. Distinct DNA fingerprints were obtained for each strain, permitting their identification. On the other hand, each strain showed little or no clonal variation. The information from the fingerprinting maps could be used for constructing phenograms and cladograms of the species and strains of Leishmania.

Animals↗
PubMed2006

Development of an efficient retrotransposon-based fingerprinting method for rapid pea variety identification.

Fast and efficient DNA fingerprinting of crop cultivars and individuals is frequently used in both theoretical population genetics and in practical breeding. Numerous DNA marker technologies exist and the ratio of speed, cost and accuracy are of importance. Therefore even in species where highly accurate and polymorphic marker systems are available, such as microsatellite SSR (simple sequence repeats), also alternative methods may be of interest. Thanks to their high abundance and ubiquity, temporary mobile retrotransposable elements come into recent focus. Their properties, such as genome wide distribution and well-defined origin of individual insertions by descent, predetermine them for use as molecular markers. In this study, several Ty3-gypsy type retrotransposons have been developed and adopted for the inter-retrotransposon amplified polymorphism (IRAP) method, which is suitable for fast and efficient pea cultivar fingerprinting. The method can easily distinguish even between genetically closely related pea cultivars and provide high polymorphic information content (PIC) in a single PCR analysis.

Breeding↗
PubMed2001

Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

Bacterial Proteins↗
PubMed1999

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines.

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

Base Sequence↗
PubMed1991

A comparative investigation and identification of Leptospira interrogans serogroup icterohaemorrhagiae strains by monoclonal antibody and DNA fingerprint analyses.

The identification of Leptospira interrogans icterohaemorrhagiae strains from a number of Reference Laboratories were confirmed using monoclonal antibody (MoAb) and DNA restriction endonuclease (EcoR1) analyses. With a few exceptions, strain fidelity was demonstrated. Three clinical isolates and one isolate from a rat (Rattus norvegicus) were identified on DNA fragment patterns and found to be similar to the reference strains, icterohaemorrhagiae copenhageni, I. "icterohaemorrhagiae" Ictero I and I. icterohaemorrhagiae RGA.

Animals↗