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PubMed1994

Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species.

The qualitative and quantitative monosaccharide spectra of purified yeast cell walls revealed that there are three phylogenetically distinct lineages of sterigma-forming basidiomycetous yeasts: (i) Kurtzmanomyces and Sterigmatomyces species, which contain high levels of mannose; (ii) Tilletiopsis species, which contain glucose, galactose, and small amounts of mannose; and (iii) Fellomyces, Kockovaella, Sterigmatosporidium, and Tsuchiyaea species, which appear to be closely related on the basis of their high levels of glucose and the presence of xylose. The yeast cell wall neutral sugars of Sporobolomyces antarcticus and Sterigmatomyces aphidis were similar to those of members of the genus Tilletiopsis. However, the possibility that these taxa are conspecific was eliminated by the results of a random amplified polymorphic DNA (RAPD) analysis. The conspecificity of Mrakia frigida and Mrakia nivalis, the conspecificity of Mrakia gelida and Mrakia stokesii, and the conspecificity of Sterigmatomyces halophilus and Sterigmatomyces indicus were confirmed by RAPD analysis results. RAPD analysis was found to be a simple and highly sensitive method which can be used to differentiate species at the DNA level; it can replace nuclear DNA-nuclear DNA hybridization experiments for species identification, characterization, and delimitation.

Base Sequence↗

PubMed2004

Genetic variability in the species Rhizopus stolonifer, assessed by random amplified polymorphic DNA analysis.

Rhizopus stolonifer is an important post-harvest pathogenic fungus. Recent taxonomic findings based on morphological and growth characteristics led to a dramatic reduction in the number of accepted species within the genus. The aim of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random amplified polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, but only low or moderate variability was observed within these groups. These results suggest a high genetic heterogeneity in the case of R. stolonifer: isolates of R. stolonifer var. stolonifer, R. stolonifer var. reflexus and R. niveus displayed species-level genetic distances, which gives rise to considerations that they might be separate species.

Base Sequence↗

PubMed1999

Characterization of Giardia lamblia groups A and B from North India by isoenzyme and random amplified polymorphic DNA analysis.

Giardia lamblia (syn. G. intestinalis) infection in young adults leads to acute/chronic diarrhea in some individuals and is asymptomatic in others. Recently, G. lamblia strains have been characterized as group A (symptomatic) and group B (asymptomatic or control) by advanced isoenzyme and molecular biology studies. In the present brief pilot study, ten G. lamblia isolates obtained from five symptomatic (group A) and five asymptomatic (group B) persons were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis. Isoenzyme analysis demonstrated remarkable homogeneity in seven enzyme patterns, the exception, being that of phosphoglucomutase, for which two zymodemes (I and III) were observed. In contrast, RAPD analysis showed homogeneity for eight primers; exceptions were two primers, A02 and B05, which separated group A G. lamblia isolates into two rapdemes (A(R1) and A(R2)) and group B G. lamblia isolates into four rapdemes (B(R1), B(R2), B(R3) and B(R4)). Further phenetic analysis showed average genetic distances of 0.105 within group A and 0.121 within group B G. lamblia isolates according to Jaccord's distance scale, which suggests that both lineages appear to consist of a range of variants with no significant (P < 0.05) genetic diversity. The two techniques demonstrated a positive association with regard to differentiation between group A and group B G. lamblia isolates. These very preliminary results indicate that RAPD analysis could be a potentially useful substitute for isoenzyme analysis.

Adolescent↗

PubMed2003

Discrimination of the closely related biocontrol agents Macrolophus melanotoma (Hemiptera: Miridae) and M. pygmaeus using mitochondrial DNA analysis.

The separation of the closely related predatory species Macrolophus melanotoma Costa (= M. caliginosus Wagner) and Macrolophus pygmaeus (Rambur) based exclusively on the different colour pattern of the first antennal segment (white central band in M. melanotoma and entirely black in M. pygmaeus) is rather precarious and their taxonomic status is still in doubt. In the present study their taxonomic status was evaluated by DNA confirmatory analysis and hybridization experiments between M. pygmaeus and a Macrolophus taxon, resembling M. melanotoma, with a first antennal segment entirely black or with a white central band collected from Dittrichia viscosa (L.) W. Greuter (Asteraceae) in southern Greece. Adult females from Dittrichia plants hybridized with males of M. pygmaeus and vice versa did not produce viable eggs. The Macrolophus species from Dittrichia irrespective of the first antennal segment coloration differed from M. pygmaeusin digestive patterns generated by AseI, XbaI, and MseI on 16S rRNA and in RAPD profiles produced by the primers OPA-18 and OPA-20. These results demonstrate that on Dittrichia plants there is a distinct dimorphic taxon, M. melanotoma, as it is the only species of the genus Macrolophus bearing a first antennal segment with a central white band. Given the limitation of the coloration pattern, the mtDNA genetic markers are the appropriate method for the identification of M. melanotomaand M. pygmaeus.

Animals↗

PubMed1998

Some new techniques for RNA or DNA analysis of interest for immunologists.

Technological advances in the field of nucleic acid analysis were the object of a recent meeting held in Lisbon. This report presents those techniques that may be most useful for immunologists as well as some of their potential applications. The topics covered include automation, quantitative PCR and its alternatives, oligonucleotide chips, and in situ hybridization and PCR.

Allergy and Immunology↗

PubMed2001

Mitochondrial DNA analysis of the putative heart of Louis XVII, son of Louis XVI and Marie-Antoinette.

According to official historiography, the 10-year-old Louis XVII died in the Temple of Paris on June 8, 1795. However, public rumour spread the theory that Louis XVII escaped and that his descendants would be alive today. One such putative 'Louis XVII' was Carl Wilhelm Naundorff, who died in 1845 in Delft (the Netherlands). Comparative mitochondrial DNA (mtDNA) analysis gave evidence that his remains could not be identified as those of Louis XVII. In the present study, mtDNA analysis was performed on the heart of the young boy who died in the prison of Paris in 1795. In order to obtain the strongest evidence possible, two laboratories independently analysed the heart. The results showed that the consensus mtDNA sequence of the heart was identical to that of the maternal relatives of Louis XVII.

DNA, Mitochondrial↗

PubMed1999

Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B.

Low-level viremia due to hepatitis B virus (HBV) was demonstrated in the sera of two patients diagnosed previously as having non-B, non-C chronic hepatitis. Both patients had a "silent" HBV infection, because they were negative for both hepatitis B surface antigen (HBsAg) and anti-hepatitis B core antibody. The TaqMan chemistry polymerase chain reaction (PCR) amplified the HBV DNA, enabling quantitation of the virus in their sera. Their serum HBV DNA concentrations were low: the amount of each HBV S or X gene amplified showed there were approximately 10(3) copies/ml and HBV DNA was detected occasionally during clinical follow-up. Positive HBsAg staining in liver tissues was demonstrated by an immunoperoxidase technique. Vertical transmission of silent HBV from one patient to her daughter was confirmed. Direct nucleotide sequencing of the amplified HBV X region revealed several mutations, suggesting reduced viral replication. One patient had a T-to-C mutation at the extreme 5'-terminus of the direct repeat 2 region and the other exhibited a coexisting X region with a 155-nucleotide deletion. These findings suggest that HBV replication is suppressed considerably in patients with silent hepatitis B.

Adult↗

PubMed2006

A comparison of individual genotyping and pooled DNA analysis for polymorphism validation prior to large-scale genetic studies.

Polymorphism validation is an important issue in genetic studies because only polymorphic markers provide useful information. We analyzed genetic data for 180 SNPs in the human major histocompatibility complex region in Caucasian and Taiwanese populations, and evaluated ethnic heterogeneity between these populations to illustrate the importance of polymorphism validation. An initial individual genotyping experiment (IGE) with 95 samples was compared with a DNA pooling allele-typing experiment (PAE) of 630 individuals for polymorphism validation based on authentic data sets. Afterwards, all samples were genotyped individually in a confirmation study. Under narrow (broad) polymorphism criteria, 24 (41) polymorphic SNPs in Caucasians could not be validated in the Taiwanese population, suggesting a 13% (23%) inconsistency rate and revealing a strong discrepancy between genetic backgrounds, probably due to ethnic heterogeneity. IGE yielded high sensitivity and specificity for polymorphism validation, but may be sensitive to sampling variation. PAE showed high sensitivity (97%) and specificity (100%) using a narrow polymorphism criterion, but reduced specificity (83%) using a broad criterion. Public domain polymorphism databases should therefore be used with caution and polymorphism validation should be performed routinely prior to conducting large-scale genetic studies. PAE is a cost-saving, reliable alternative to IGE for polymorphism validation, especially for a stringent polymorphism criterion.

Asian People↗

PubMedDate not supplied

The use of dynamic size-sieving capillary electrophoresis and mismatch repair enzymes for mutant DNA analysis.

The detection of base pair mismatches in limiting amounts of DNA is important in the early diagnosis of cancer and other genetic diseases. The specific type and exact location of a bp mismatch in certain genes can yield information on the likelihood of a patient developing a genetic disease, as well as the severity of the disease. We demonstrate two methods of specific DNA point mutation detection and identification that involve the integration of mismatch repair cleavage enzyme analyses with dynamic size-sieving capillary electrophoresis. The mismatch repair cleavage enzymes employ the very mechanism that a cell uses in its own mismatch recognition and repair systems. One analysis employs an isothermal signal amplification process, and the other involves polymerase chain reaction (PCR) (Hoffmann-LaRoche, Nutley, NJ, U.S.A.) for amplification of the DNA. Separation of the DNA fragments using dynamic size-sieving CE yields a cleaved fragment, providing definitive evidence of a bp mismatch. The specificity and sensitivity of the assay are facilitated by the detection of fluorescently labeled DNA fragments using laser-induced fluorescence detection; picogram quantities of a target DNA can be analyzed reproducibly.

Base Pair Mismatch↗

PubMed2001

Characterization of psychrotrophic bacteria in the surface and deep-sea waters from the northwestern Pacific Ocean based on 16S ribosomal DNA analysis.

Seventy-eight 4 degrees C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0-200 m) and deep-sea (1000-9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4 degrees C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats.

Journal Article↗

PubMed1998

Application of random amplified polymorphic DNA analysis for detection of Salmonella spp. in foods.

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 x 10(3) cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 x 10(-1) CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42 degrees C. Seven raw foods inoculated with S. typhimurium at numbers from 4 x 10(-1) to 2.6 x 10(2) CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.

Base Sequence↗

PubMed1990

Flow cytometric DNA analysis of lesions from 18 children with langerhans cell histiocytosis (histiocytosis x).

The DNA content in 26 formalin-fixed, paraffin-embedded histologic specimens from 18 children with Langerhans cell histiocytosis (LCH) was analyzed by flow cytometry. In two cases, the propidium iodide fluorescence histograms showed small (5 and 3% of the analyzed nuclei) but significant aneuploid subpopulations with DNA indices of approximately 1.5. This was confirmed by analysis of unfixed, frozen material in one patient. Both patients had disseminated disease without organ dysfunction and were treated with prednisone. Currently, they are without signs of disease activity after 1 and 10 years. DNA histograms were normal from a patient who died from disseminated disease and from two patients with disseminated disease who experienced several relapses and various chemotherapeutical regimens. The histograms were also normal in lesions from four patients with unifocal bone involvement. Our results show that DNA aneuploidy occurs in LCH lesions in the pediatric age group. Further investigation is necessary to reveal whether DNA aneuploidy is restricted to disseminated LCH or its presence has any value in predicting the course and outcome of the disease.

Adolescent↗

PubMed1997

Hb T-Cambodia, a beta chain variant with the mutations of Hb E and Hb D-Punjab, confirmed by DNA analysis.

Hemoglobin variants with two amino acid substitutions affecting one globin chain are relatively rare. Hb T-Cambodia, a doubly substituted beta-globin variant, was characterized previously by amino acid sequencing as having sequence alterations in beta 26 (beta 8)Glu-->Lys and beta 121(GH4) Glu-->Gln (1). It is a variant that migrates cathodic to Hb A2 on alkaline electrophoresis and with Hb A on acid citrate agar electrophoresis. We report here the mutations of Hb T-Cambodia at the nucleotide level using DNA sequencing, in beta-globin gene codon 121 (GAA-->CAA) and in codon 26 (GAG-->AAG). These are the mutations of Hb D-Punjab and Hb E, respectively.

Adult↗

PubMed1997

DNA analysis indicates patient-specific human papillomavirus type 16 strains in Bowen's disease on fingers and in archival samples from genital dysplasia.

Human papillomavirus (HPV) type 16 is casually involved in the pathogenesis of anogenital cancer and has also been demonstrated in some patients with Bowen's disease (BD) on the fingers. From two women with HPV 16 in BD on the fingers, and in archival samples from genital dysplasia, collected as long as 26 years ago, the non-coding region of the virus was amplified by the polymerase chain reaction and sequenced. The HPV 16 DNA sequences found in the finger lesions and in the genital archival samples showed no diversities within single patients. Compared with an HPV 16R reference sequence, one patient showed a unique T nucleotide at position 78, whereas the other patient exhibited T and A nucleotides at positions 7193 and 7521, respectively. In one of the patients, the same strain of HPV 16 was found in a digital tumour 26 years after its clearance from the genital tract. DNA sequence analysis indicated patient-specific HPV 16 strains. Auto-inoculation from the genital tract was favoured as a plausible explanation of why HPV 16 caused BD on the fingers.

Adult↗

PubMed2005

Mitochondrial DNA analysis of the bark beetle predator Thanasimus dubius F. (Coleoptera: Cleridae) reveals regional genetic differentiation.

The checkered beetle, Thanasimus dubius F., is an important predator of scolytid bark beetles that attack conifers. Relatively few studies exist that have addressed the population genetics of predatory beetles, especially those with potential as biological control agents. This study was conducted to investigate the population genetics of T. dubius across a large part of its range in the eastern United States. A 464-base pair portion of the mitochondrial cytochrome c oxidase subunit I was sequenced for 85 individuals resulting in 60 haplotypes. Analysis of molecular variance was conducted on the resulting haplotypes for all populations and as a hierarchical analysis between populations defined as broad-scale northern and southern groups. Results indicate a significant overall Phi ST = 0.220 (P < 0.001) for all populations with the hierarchical analysis revealing that this significant Phi ST is due to structuring of the populations between northern and southern regions (Phi CT = 0.388, P < 0.009). The observed genetic structure is possibly due to the discontinuous distribution of pine trees, which act as hosts for the prey of T. dubius, which has occurred historically in the central region of the United States that has been covered by prairie.

Animals↗

PubMed1986

Retrospective DNA analysis of head and neck squamous cell carcinoma.

Abnormal cellular DNA content, a hallmark of malignancy, is known to occur in squamous cell carcinoma of the head and neck. The significance of this finding has yet to be elucidated. Paraffin-embedded blocks from 46 squamous cell carcinomas of the head and neck were sectioned, dewaxed, and made into a suspension of bare nuclei by mechanical mincing and enzymatic digestion. The nuclei were stained with propidium iodide, and quantitative DNA measurements were obtained with flow cytometry. The results were compared with existing prognostic factors: DNA aneuploidy was identified in 41% of the specimens and was significantly correlated with tumor size and surgical stage; the relationship between DNA aneuploidy and anatomic site approaches statistical significance. There was no correlation with histologic grade or presence or absence of cervical metastasis.

Aneuploidy↗

PubMed2002

Assessment of genomic instability in breast cancer and uveal melanoma by random amplified polymorphic DNA analysis.

Some types of cancer have been associated with abnormal DNA fingerprinting. We used random amplified polymorphic DNA (RAPD) to generate fingerprints that detect genomic alterations in human breast cancer. Primers were designed by choosing sequences involved in the development of DNA mutations. Seventeen primers in 44 different combinations were used to screen a total of 6 breast cancer DNA/normal DNA pairs and 6 uveal melanoma DNA/normal DNA pairs. Forty-five percent of these combinations reliably detected quantitative differences in the breast cancer pairs, while only 18% of these combinations detected differences in the uveal melanoma pairs. Fourteen (32%) and 12 (27%) primers generated a smear or did not produce any band patterns in the first and second cases, respectively. Taking into account the ability of RAPD to screen the whole genome, our results suggest that the genomic damage in breast cancer is significantly higher than in uveal melanoma. Our study confirms other reports that the molecular karyotypes produced with random priming, called amplotypes, are very useful for assessing genomic damage in cancer.

Breast Neoplasms↗

PubMed1989

Angiomatoid malignant fibrous histiocytoma: flow cytometric DNA analysis of six cases.

Using flow cytometry, we examined the DNA content of six angiomatoid "malignant" fibrous histiocytomas (AMFH), seven nonangiomatoid MFH, and seven myxoid variants of MFH. All six AMFHs were diploid. All nonangiomatoid MFHs and six of seven of the myxoid variant were aneuploid. The clinical course, histological appearance, and ploidy pattern of AMFH are distinctly different from other MFH variants. These differences strongly suggest that AMFH is at the benign end of the clinicobiologic spectrum manifested by "malignant" fibrous histiocytomas.

Adolescent↗