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At least 397 records · Page 22Linked to original sources
PubMed1997

Characterization of Scedosporium prolificans clinical isolates by randomly amplified polymorphic DNA analysis.

Fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate Scedosporium prolificans isolates. A total of 59 arbitrary primers were screened with six unrelated S. prolificans isolates, and a panel of 12 primers was selected. The 12 primers were then used to detect DNA polymorphisms among 17 S. prolificans isolates from 11 patients with systemic S. prolificans infections diagnosed in three hospitals located in geographically different areas of Spain. Eight patients were diagnosed with S. prolificans infection in a single institution over a 6-year period, and two other patients were diagnosed with S. prolificans infection in a different hospital over a 1-year period. No single primer allowed for the discrimination of all the isolates from different patients, but this was possible by combining the RAPD patterns from three primers (UBC 701, AB1.08, and AB1.11 or UBC 701, AB1.08, and UBC 707). However, multiple isolates from the same patient were identical. In this study, we also compared a visual method and a computerized method for the analysis of the RAPD patterns. Both methods were satisfactory and gave few discordances, but given the advantages and disadvantages of each method, both systems should be used together. RAPD analysis provided a fast and economical means of typing S. prolificans isolates, with a high level of discrimination among unrelated isolates. Typing by RAPD analysis confirmed that the S. prolificans infections were epidemiologically unrelated.

DNA Primers↗
PubMed2004

Molecular-beacon-based array for sensitive DNA analysis.

Molecular beacon (MB) DNA probes provide a new way for sensitive label-free DNA/protein detection in homogeneous solution and biosensor development. However, a relatively low fluorescence enhancement after the hybridization of the surface-immobilized MB hinders its effective biotechnological applications. We have designed new molecular beacon probes to enable a larger separation between the surface and the surface-bound MBs. Using these MB probes, we have developed a DNA array on avidin-coated cover slips and have improved analytical sensitivity. A home-built wide-field optical setup was used for imaging the array. Our results show that linker length, pH, and ionic strength have obvious effects on the performance of the surface-bound MBs. The fluorescence enhancement of the new MBs after hybridization has been increased from 2 to 5.5. The MB-based DNA array could be used for DNA detection with high sensitivity, enabling simultaneous multiple-target bioanalysis in a variety of biotechnological applications.

Base Sequence↗
PubMed1999

[Forensic DNA analysis--past and future].

Since the introduction of DNA polymorphism analysis techniques to forensic science, forensic identification research has made radical, astonishing progress at a rate that has already rendered the initial methodologies introduced fifteen years ago obsolete. DNA extraction now can be quickly and efficiently performed by various kinds of commercially available kits. The advent of PCR has enabled the use of relatively crude and minute DNA as amplification templates while many kinds of new detection methods for analyzing the amplified products have also been developed. Although many minisatellites such as MCT118, YNZ22, COL2A1, and ApoB were highlighted at the beginning of 1980s, none of these loci, with the exception of MCT118, have proved useful for forensic DNA application due to their low amplification efficiency. On the other hand, STR loci containing four base pair repeat sequences have been used routinely for human identification since the mid-1990s. In the near future, the highly efficient STR should be selected as a consensus core marker in Japan. STR systems located on the Y chromosome are widely used in forensic science for the identification of male individuals. These systems have a special significance in forensic science cases where mixtures of male and female DNA are analyzed, as happens in cases of rape or other sexual crimes. The characteristics of high copy number, maternal inheritance, and high degree of sequence variability make mtDNA a powerful tool for forensic identification. Most of the variations in mtDNA among individuals are found within the displacement loop (D-loop). In all population groups, mtDNA sequences can be useful for discriminating among unrelated individuals. Now it is necessary to get as much as possible individual genetic information as quickly as possible in order to enable individual identification. We will create a new era in which forensic identification can be performed using microarray technology.

Blotting, Southern↗
PubMed2006

Image cytometric DNA analysis of mucosal biopsies in patients with primary achalasia.

AIM: To determine DNA aneuploidy in mucosal biopsies of achalasia patients for subsequent rapid diagnosis. METHODS: Biopsies from the middle third of the esophagus were obtained in 15 patients with achalasia. Immunohistochemical staining was carried out with monoclonal antibodies MIB-1 for Ki67 and PAb 1801 for p53, in addition to the conventional histologic examination for dysplasia. Nuclei of fresh biopsy material were enzymatically and mechanically isolated, and the DNA content was determined with image cytometry after Feulgen staining. DNA grading of malignancy was assessed according to Boecking to determine the variability of DNA values noted around the normal diploid peak. Further indices measured included the aneuploid rate, and the 5c-, 7c- and 9c-exceeding rate. RESULTS: The histological examination did not demonstrate dysplasia; while MIB-1 (basal) showed a positive reaction in 8/15 achalasia specimens, p53 was negative in all specimens. Image cytometric DNA analysis detected aneuploidy in 4/15 (26.7%) specimens. Samples from 15 patients with squamous cell carcinoma as well as specimens obtained exclusively 2 cm proximal to the tumor served as reference tests. All carcinomas (15/15) as well as 9 of the peritumoral samples (9/15) were aneuploid. The comparison of biopsies from achalasia patients with peritumoral and carcinoma specimens revealed statistically significant differences regarding the aneuploid rate (diploid: P < 0.0001; tetraploid: P = 0.001), grading of malignancy according to Boecking (P < 0.0001) and the 5c- (P < 0.0001), 7c- (P < 0.0001), and 9c- (P = 0.0001) exceeding rate with progredient DNA alterations in the respective order. CONCLUSION: The finding that DNA aneuploidy was identified by image cytometry in esophageal specimens of patients with achalasia, which may be due to specific chromosomal alterations presenting as precancerous lesions in 27% of patients, leads us to conclude that image cytometry represents a valuable screening tool.

Adult↗
PubMed1992

Phylogeny of the genera Trichophyton using mitochondrial DNA analysis.

Diversity of mitochondrial DNA (mtDNA) was investigated in 92 Trichophyton rubrum strains, 2 T. mentagrophytes var. mentagrophytes, 2 T. m. var. interdigitale, 2 T. m. var. goetzii, 1 T. m. var. erinacei, 2 T. quinckeanum, 2 T. schoenleinii, 1 T. tonsurans, 2 T. verrucosum var. album, 2 T. v. var. discoides, 1 T. violaceum var. violaceum, 1 Arthroderma benhamiae, and 1 A. vanbreuseghemii using endonucleases, Hae III, Msp I, Hind III, Xba I, and Bgl II. Trichophyton species were divided into 7 groups, and a phylogenetic tree was produced based on sequence divergence within mtDNA. The following results were obtained: (1) T. rubrum was divided into 2 groups Type I and Type II, and was suggested to be a complex. (2) A. benhamiae was closely related to T. m. var. erinacei. (3) T. rubrum Type II, T. tonsurans, and A. vanbreuseghemii showed identical restriction profiles, and were suggested to be closely related to each other or identical. (4) T. quinckeanum and T. schoenlenii showed identical restriction profiles, which differed slightly from those of A. vanbreuseghemii. (5) mtDNA analysis was useful in identifying pleomorphic strains.

DNA, Fungal↗
PubMed1995

Flow cytometric DNA analysis of classic and steroid-induced Kaposi's sarcoma.

Flow cytometric DNA analysis of various tumours has indicated a correlation between the degree of malignancy and ploidy; results which could have clinical significance. We analysed the ploidy of Kaposi's sarcoma (KS) tumours, and classified the results according to clinical history and histological findings. We found that patients on steroid treatment had an aneuploid pattern, and most of the patients with classic-type KS had a diploid pattern on flow cytometry.

Aged↗
PubMed1995

[DNA analysis in heterozygotes in familial hypercholesterolemia].

BACKGROUND: Accuracy of clinical diagnosis of heterozygotes with familial hypercholesterolemia (FH) is limited. The aim of our study was to demonstrate possibilities of progressive diagnostic approach, DNA analysis, LDL receptor gene (LDLR) and apolipoprotein B (ApoB) in case of our study, and compare our results with the data obtained in other populations. METHODS AND RESULTS: The low density lipoprotein receptor (LDLR) gene RFLP frequencies for restriction endonucleases AvaII, HincII, NcoI, PvuII and StuI were determined in the sample of 52 FH patients and in the group of 37 healthy individuals. Using PCR, the LDLR gene was then tested for Pro664-Leu and Val408-Met point mutations. The first DNA diagnosis of familial defective apolipoprotein B-100 (FDB) using point mutation PCR analysis of 26. exon of ApoB gene in Czech Republic was performed. LDLR gene RFLP frequencies for restriction endonucleases AvaII, HincII, NcoI, PvuII and StuI in he sample of 52 FH patients were 0.48, 0.52, 0.73, 0.31 and 0.93 respectively. LDLR gene RFLP frequencies for enzymes AvaII, HincII, NcoI, PvuII and StuI in the group of 37 healthy individuals were 0.39, 0.50, 0.70, 0.22 and 0.99 respectively. In the group of FH patients no point mutations Pro664-Leu and Val408-Met were detected. However, there was found 110bp insertion in the 9th exon of LDLR gene in two FH patients during studies of Val408-Leu mutation. Two FDB probands in the FH group and another 7 FDB individuals in probands families were detected. FDB frequency in the sample of FH patients was 3.8%. CONCLUSIONS: LDLR gene RFLP frequencies and FDB frequency in our group of FH patients did not differ from that of FH patients in other Caucasian populations. DNA analysis is advantageous complementary method for diagnosis of FH and is irreplaceable for the detection of FDB.

Adult↗
PubMed1984

DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy.

DNA analysis of small insect iridovirus 6 was performed. Combined exonuclease-restriction endonuclease digestions revealed that all resulting fragments were degraded without preference for any one DNA fragment. Upon denaturation and reannealing of native linear Chilo iridescent virus DNA (158 x 10(6) daltons), duplex DNA circles of a smaller size (140 x 10(6) daltons) with protruding tails were formed.

Journal Article↗
PubMed2004

Genetic substructuring within Oesophagostomum bifurcum (Nematoda) from human and non-human primates from Ghana based on random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) was used to study genetic variation within Oesophagostomum bifurcum in Ghana. Four different decamer primers were used for the amplification of DNA from individual O. bifurcum adults (n = 41) from humans and non-human primates (including the Mona monkey, Patas monkey and Olive baboon) from different geographic regions. Analysis of the amplicons from all 41 nematodes by high resolution, denaturing polyacrylamide gel electrophoresis defined a total of 326 informative RAPD bands. Cluster analysis of the RAPD data (based on pairwise comparison of banding profiles) showed that O. bifurcum from humans was genetically distinct from O. bifurcum from the Mona and Patas monkeys, and from the Olive baboon. These findings clearly demonstrate the existence of population genetic substructuring within O. bifurcum from different primate hosts in Ghana, and raise interesting questions about host specificity, epidemiology (e.g., zoonotic transmission), and ecology of the different genotypes of O. bifurcum.

Adolescent↗
PubMed1998

Virtual DNA analysis--a new tool for combination and standardised evaluation of SSO, SSP and sequencing-based typing results.

In order to obtain reliable information on HLA types, DNA typing with sequence-specific oligonucleotide/primer (SSO/SSP) typing sets or sequencing-based typing (SBT) is increasingly performed. The quality of the evaluation depends on the presence of a complete listing of all typed alleles as well as on the ability of detecting all corresponding alleles/allele pairs. We have developed the concept of virtual DNA analysis (VDA), which is able to combine all types of SSO/SSP/SBT results and evaluate this typing in combination according to the latest published allele sequence lists. The concept is based on the target DNA recognised by the respective typing techniques. All SSO/SSP or SBT results are transformed to a virtual sample DNA, which subsequently is analysed. Evaluation of generic or allele-specific DNA typing or the combination of both is supported. Due to this flexible approach, all kinds of SSO/SSP sets, as far as the respective SSO/SSP sequences are available, can be entered and evaluated immediately. The combination of collected data of different typing sets and procedures leads to the highest possible typing resolution. If more than one possible allele combination persists, the program reduces the result to the most specific common denominator in a stepwise manner. VDA offers the possibility of re-evaluation of former SSO/SSP/SBT results, alone or in combination. No solutions are omitted. This might be a first step towards standardisation of evaluating DNA-based HLA typing results or transfer of the respective typing data for later evaluation.

Databases, Factual↗
PubMed2002

Determining the genetic structure of the natural population of Staphylococcus aureus: a comparison of multilocus sequence typing with pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing.

We used a sample of Staphylococcus aureus strains that are carried by humans and that are representative of the natural population of S. aureus strains in order to assess the value of multilocus sequence typing (MLST), pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing as epidemiological tools. Only MLST was able to define clonal complexes unambiguously. All DNA-based typing approaches achieved a high degree of agreement, implying phylogenetic concordance, but predicted epidemiological associations with variable accuracy.

Bacterial Typing Techniques↗
PubMed1997

The applicability of formalin-fixed and formalin fixed paraffin embedded tissues in forensic DNA analysis.

Historically, formalin fixed (FF) tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction (PCR) technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study evaluates the potential use of DNA from FF and formalin fixed paraffin embedded (FFPE) tissues in 13 PCR systems; HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, GC, D1S80, vWA31, THO1, F13A1, FES/FPS, TPOX, and CSF1PO. The first six, HLA DQ alpha, LDLR, GYPA, HBGG, D7S8, and GC are reverse dot blot systems, D1S80 is an amplified fragment length polymorphism (AmpFlp) system and the others are short tandem repeats (STRs). This study shows that FFPE tissue which has not been fixed in formalin for more than three days is a useful source of DNA for 12 of the 13 PCR systems. In contrast, FF tissue did not prove to be a reliable source of DNA for the PCR techniques examined here.

Chelating Agents↗
PubMed1985

A study of enterotoxigenic Escherichia coli, serogroup 0126, by bacterial restriction endonuclease DNA analysis (BRENDA).

Sixteen isolates of Escherichia coli were subjected to bacterial restriction endonuclease DNA analysis (BRENDA). Nine of these isolates were from an outbreak of human diarrhoea and produced stable toxin, the remaining seven were non-toxigenic strains from animal and human sources. The isolates from the outbreak produced indistinguishable DNA electrophoretic patterns in spite of their assignment to seven different H serotypes. Their BRENDA patterns were markedly different from the other isolates examined. These results support the epidemiological evidence that a single-strain outbreak had occurred, and they cast doubt on the value of H typing for this particular investigation.

Bacterial Toxins↗
PubMed2003

High-resolution single-stranded DNA analysis on 4.5 cm plastic electrophoretic microchannels.

Plastic microchannels (4.5 cm long) fabricated from an etched glass master were tested for high-resolution single-stranded DNA analysis. Using replaceable denaturing linear polyacrylamide as sieving matrix, one-color separation of a fragment sizing standard with single-base resolution (R > 0.5) was achieved up to 275 bases. Two-color sizing analysis of four loci short tandem repeat (STR) allelic ladder (CSF1PO, TPOX, TH01, vWA) with single-base resolution (R = 0.62) on TH01 alleles 9.3 (198 bp) and 10 (199 bp) was demonstrated. An average standard deviation of +/- 0.06 bp and +/-0.11 bp in sizing 32 alleles of the CTTv ladder was attained between runs and between channels, respectively. Four-color sequencing separation of a terminator sequencing standard showed a base-calling accuracy of 99.1% out to 320 bases in 13 min.

Acrylic Resins↗
PubMed1991

Renal oncocytoma: long-term follow-up and flow cytometric DNA analysis.

We report a retrospective study on the clinicopathologic features and flow cytometric DNA analysis of ten renal oncocytomas compared with a control group of ten randomly selected renal cell carcinomas. Among the oncocytoma patients, no recurrences or metastases were noted over an average follow-up of 6.7 years (range = 6 months to 16 years). Reproducible, high-quality DNA histograms were obtained on the paraffin-embedded specimens by using our modified flow cytometric procedure. One aneuploid (10%) and two hyperdiploid tumors (20%) were found in the oncocytoma group. There was no correlation between these abnormal DNA histographic patterns and survival or tumor stages. On the contrary, a good correlation was found between tumor grades and DNA ploidy in the controls. We conclude that renal oncocytoma is a clinically benign tumor, yet it may exhibit varying degrees of flow cytometric DNA abnormalities, which have no predictive value on survival and probably reflect the characteristics of oncocytes rather than its malignant potential.

Adenoma↗