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PubMed1992

Mitochondrial DNA analysis in Leber's hereditary optic neuropathy.

A mitochondrial DNA (mtDNA) mutation at nucleotide 11778 has been reported as the genetic defect associated with Leber's hereditary optic neuropathy (LHON), but some pedigrees failed to reveal this mutation. The authors present the genetic analysis of a large Italian LHON family with three probands and 16 asymptomatic maternal relatives. The 11778 mtDNA mutation was present in this family and absent in 52 Italian healthy controls, confirming the association between this genetic defect and LHON. In addition, they found a variable proportion of mutated and wild-type mtDNA (heteroplasmy) in every maternally related family member, including the probands. Different patterns of heteroplasmy were present in the pedigree and lower levels of wild-type mtDNA seemed to correlate with the disease status. Moreover, the authors evaluated the mitotic segregation of mt genomes in blood, hair and urinary tract epithelia of the three patients and they found a similar level of heteroplasmy in these tissues.

Base Sequence↗
PubMed1996

Comparative DNA analysis of Bordetella pertussis clinical isolates by pulsed-field gel electrophoresis, randomly amplified polymorphism DNA, and ERIC polymerase chain reaction.

We used DNA fingerprinting by pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) and PCR amplification of enterobacterial repetitive intergenic consensus sequences (ERIC-PCR) to compare 15 clinical isolates of Bordetella pertussis recovered between August 1993 and September 1995 from 13 infants and two adults, living in the same geographic area. PFGE produced 10 patterns and made it possible to differentiate all the isolates and to indicate an intrafamilial transmission. RAPD and ERIC-PCR generated banding patterns with small differences and had a poor discriminatory power. During the last 2 years, at Armand-Troussau pediatric hospital, 10 distinct clones of clinical B. pertussis isolates, with a predominant clone including seven strains, could be determined by the PFGE method.

Adult↗
PubMed1995

Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis.

Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we observed a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 microM tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 microM dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants.

Animals↗
PubMed2006

Random amplified polymorphic DNA analysis of Campylobacter jejuni and Campylobacter coli isolated from healthy cattle and sheep.

The genetic heterogeneity among Campylobacter jejuni and Campylobacter coli isolates obtained from apparently healthy cattle and sheep was investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 348 Campylobacter isolates, consisting of C. jejuni (n = 218) and C. coli (n = 130), were analysed. All these isolates were successfully typed by RAPD analysis. The total numbers of band patterns defined by RAPD in cattle and sheep were 42 and 45, respectively. Of the 42 distinct types obtained from cattle, 37 types were observed in C. jejuni isolates (n = 115), and the remaining 5 were in C. coli isolates (n = 30). Of 45 distinct types obtained from sheep, 21 types were observed in C. jejuni isolates (n = 103), and 24 were in C. coli isolates (n = 100). It was concluded that a high degree of heterogeneity existed among the C. jejuni and C. coli isolates of healthy cattle and sheep.

Animals↗
PubMed2004

Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry.

We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP-PC-Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5-position of 2'-deoxyribouridine 5'-triphosphate via a photocleavable 2-nitrobenzyl linker. We have demonstrated that dUTP-PC-Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP-PC-Biotin can be efficiently isolated by a streptavidin-coated surface and recovered by near-UV light irradiation at room temperature in mild condition for further analysis without using any chemicals or heat. Single and multiple primer extension reactions were performed using the dUTP-PC-Biotin to generate DNA products for MALDI-TOF MS analysis. Such nucleotide analogs that carry a biotin and a photocleavable linker will allow the isolation and purification of DNA products under mild conditions for MS-based genetic analysis by DNA sequencing or multiplex single nucleotide polymorphism (SNP) detection. Furthermore, these nucleotide analogs should also be useful in isolating DNA-protein complexes under non-denaturing conditions.

Biotin↗
PubMed2001

Identity of Cypripedium calceolus (Orchidaceae) in Rebun Island: comparative DNA analysis of related species.

Cypripedium calceolus was found in 1980 in Rebun Island off the north coast of Hokkaido, Japan, but the origin of this plant has been a controversial issue. In this study, we have made a comparative study by chloroplast DNA sequencing analysis among C. calceolus which occurs in Rebun Island and populations of C. calceolus from western Europe, China and far eastern Russia (Nakhodka), and also as references, C. macranthos in Japan and other Cypripedium species in North America. A Cypripedium cf. "calceolus", found recently in eastern Hokkaido, was also included in this analysis. The C. calceolus samples analyzed were categorized into three groups, i.e., those from Western Europe, from China and far eastern Russia, and from Rebun Island. The C. calceolus in Rebun Island was clearly different from the others in terms of DNA sequence and morphological features. The C. cf. calceolus from eastern Hokkaido and one sample from Nadhodka, Russia, were also classified into the same group as those from Rebun Island, although some differences in their morphological features were observed. It is concluded that the C. calceolus found in Rebun Island is not identical with those growing in Europe and China. In addition, it was found that it may be possible to classify C. macranthos into two groups, namely groups which include or do not include var. rebunense. An unidentified Cypripedium species found in Rebun Island falls into the same group as var. rebunense.

Base Sequence↗
PubMed1991

Mitochondrial DNA analysis of Leber's hereditary optic neuropathy.

A mitochondrial DNA (mtDNA) mutation associated with Leber's hereditary optic neuropathy (LHON) was recently observed. The presence or absence of the mutation was analyzed in 10 Japanese patients whose clinical course and fundus findings were consistent with LHON. Four of them had at least one maternally related individual who also had bilateral optic atrophy, and were diagnosed as "definite LHON". The other 6 cases lacked any record of optic nerve disease in maternally related individuals, and were diagnosed as "possible LHON". We found the mutation at the SfaNI site of mtDNA in 3 out of the former 4 cases, and in 5 out of the latter 6 cases. This result demonstrates the clinical and diagnostic importance of mtDNA analysis, especially with possible cases of LHON, and suggests that an alternative mutation associated with LHON is also present in Japanese patients.

Adolescent↗
PubMed1990

Mitochondrial DNA analysis in Parkinson's disease.

The reduced form of nicotinamide adenine dinucleotide coenzyme Q reductase (complex I) activity has recently been shown to be deficient in the substantia nigra of patients dying with Parkinson's disease. This biochemical defect is identical to that produced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which also produces parkinsonism in humans. Complex I comprises 25 polypeptides, seven of which are encoded by mitochondrial DNA. Restriction fragment analysis of substantia nigra DNA from six patients with Parkinson's disease did not show any major deletion. In two cases, there were different novel polymorphisms that were not observed in control brain (n = 6) or blood (n = 34) samples.

DNA Damage↗
PubMed1998

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↗