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PubMed2006

Multi-nanopore force spectroscopy for DNA analysis.

The need for low-cost DNA sequence detection in clinical applications is driving development of new technologies. We demonstrate a method for detection of mutations in a DNA sequence purely by electronic means, and without need for fluorescent labeling. Our method uses an array of nanopores to perform synchronized single-molecule force spectroscopy measurements over many molecules in parallel, yielding detailed information on the kinetics of hundreds of molecule dissociations in a single measurement.

Mutation↗
PubMed2005

Cloneless genomic DNA analysis: an efficient and simple methods for de novo genomic sequencing projects and gap filling.

The utility of using genomic DNA directly in agarose, i.e. cloneless libraries, in place of large clone libraries, radiation hybrid panels, or chromosome dissection was demonstrated. The advantage of the cloneless library approach is that, in principle, a targeted genomic resource can be developed rapidly for any genomic region using any genomic DNA sample. Here, a human chromosome 20 Not I fragment library was generated by slicing a pulsed field gel lane containing fractionating Not I cleaved DNA from a monosomic hybrid cell line into 2 mm pieces. A reliable PCR method using agarose embedded DNA was developed. InterAlu PCR generated unique patterns of products from adjacent slices (e.g. fractions). Further, the specificity of the interAlu products was demonstrated by FISH analysis and in other hybridization experiments to arrayed interAlu products. STS content mapping was used to order the fractions and also demonstrate the unique content of the library fractions.

Chromosome Mapping↗
PubMed2000

[Application of DNA analysis for differential diagnosis of familial hypercholesterolemia and familial defect of apolipoprotein b-100].

AIM: To determine occurrence of apolipoprotein B-100 mutation in codon 3500 (apoB3500) in patients with primary hypercholesterolemia in Russia. MATERIALS AND METHODS: The study included 71 patients with clinical diagnosis of familial hypercholesterolemia (FH) and 24 relatives. All the subjects were tested for the presence of apoB3500 mutation using polymerase chain reaction and cleavage with restriction enzyme HhaII. Samples demonstrating anomalous pattern were further analysed by automatic DNA sequencing. RESULTS: Apob3500 mutation was detected in two (2.8%) female patients. In both cases cholesterol levels were severely increased although clinical features were different. CONCLUSION: Some cases of primary hypercholesterolemia in Russia may be due to familial defective apoB-100. Further screening of FH patients is required for a precise estimation of the incidence rate of familial defective apoB-100 in this country.

Adult↗
PubMed1990

[Sex identification by analysis of DNA extracted from hard tissues].

Sex identification of unidentified two bleached skeletons by the method of DNA analysis in addition to the morphological examination was performed. Bones and teeth were taken out for the DNA analysis, and cracked to compounds by liquid nitrogen. DNA was extracted by the standard procedure. We used two methods for sex identification by DNA analysis; one is Southern blot hybridization with Y-chromosome specific probe (PHY10; 3.4kb), and the other is PCR (polymerase chain reaction) amplifying with sex chromosome specific fragments (alphoid satellite family). These two methods enable us to identify the sex of the two cases presented here more correctly with sufficient evidence than depending only on morphological examination. With some technical difficulties, it is possible to put DNA analysis including Southern blot hybridization or PCR into practical determination of sexing of hard tissues such as a piece of bone or a tooth, and old samples. Therefore, they seem to be effective methods applicable to forensic science.

Adolescent↗
PubMed1991

DNA analysis of oral leukoplakia by flow cytometry.

DNA ploidy of 19 oral leukoplakias with and without epithelial dysplasia was investigated and the results were compared with 11 normal gingival biopsies, 14 oral benign tumours and 50 oral squamous cell carcinomas. The results suggest a possible relationship between DNA aneuploidy and oral leukoplakias or squamous cell carcinomas, as 32% of the oral leukoplakias and 48% of the oral squamous cell carcinomas were aneuploid although all the normal gingival biopsies and the benign oral tumours examined were diploid. No significant relationship was observed, however, between DNA ploidy and epithelial dysplasia in the leukoplakias.

Adult↗
PubMed1988

DNA analysis by flow cytometry.

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.

Animals↗
PubMed2001

Quantitative nested PCR for human cytomegalovirus DNA: analysis using NucleoScan 2001.

A quantitative nested DNA PCR for human cytomegalovirus (CMV) was developed using PCR mimic DNA as an internal standard. A combination of ultra-thin gel and enhanced detection of ethidium bromide-stained PCR bands allowed us to achieve a 25-fold increase in sensitivity over the conventional gel electrophoreses, down to 0.1 ng of DNA per band. When the technique was applied to measuring CMV load in CMV positive leucocyte DNA preparations its sensitivity spanned the range from single to hundreds of CMV DNA copies in 0.1 microg of total white blood cell DNA.

Cytomegalovirus↗
PubMed1992

[Diagnosis of Lepore hemoglobinopathy with DNA analysis].

We illustrate the application of recombinant DNA methods (namely Southern blotting) for the genotype diagnosis of haemoglobin Lepore Boston: the use of the restriction endonucleases PstI and XbaI along with a beta globin gene specific probe make it possible to detect a deletion of approximately 7kb which typically characterizes the delta beta Boston gene. The impact of using such methods in the prenatal diagnosis of major haemoglobinopathies is discussed.

Adult↗
PubMed1991

Flow cytometric DNA analysis of primary cutaneous malignant melanoma.

DNA-ploidy of primary malignant melanoma was retrospectively compared by flow cytometry with sections of paraffin-embedded blocks, among tumours at different histopathological stages (levels) and among tumours and their peripheral lesion. In none of 10 melanomas at levels I-II or 10 melanocytic nevi was an aberrant DNA pattern found. In contrast, 8 of 18 melanomas at levels III-V including 6 of 9 melanomas at level V, showed a non-diploid (aneuploid) DNA pattern. However, even in these non-diploid melanomas the peripheral area showed a diploid pattern where tumour cell invasion was slight, similar to the level I-II melanomas. These results indicate that DNA-aneuploidy in melanoma is closely associated with the lesional advancement, and reflect a poor prognosis for patients with melanoma at level III or higher.

Adolescent↗
PubMed1982

Interaction of a DNA-binding protein, the gene product of D5 of bacteriophage T5, with double-stranded DNA. Analysis by metrizamide gradient centrifugation.

Interactions of DNA and the gene product D5 (gpD5) of bacteriophage T5, a DNA-binding protein that binds preferentially and cooperatively to double-stranded DNA, were analyzed by metrizamide gradient centrifugation. Conditions were set so that DNA and DNA protein complex sedimented to apparent equilibrium positions. DNA has a buoyant density of 1.12 g/cm3, and DNA saturated with gpD5 has a buoyant density of 1.17 g/cm3. These values are independent of DNA size and base composition in the range studied. At gpD5 concentration below the saturation value in a low ionic strength buffer, DNA distribution is bimodal, indicating cooperative binding of gpD5 to DNA. However, in the presence of 10 mM MgCl2, the binding process becomes distributive, with the buoyant density increasing linearly with the amount of gpD5 added until the saturation. From these data, one molecule of gpD5 is calculated to cover 40 base pairs at saturation. The technique as described has general applicability to the study of any interaction between DNA and dNA-binding proteins that bind in sufficient amount to cause detectable changes in buoyant density.

Centrifugation, Density Gradient↗
PubMed2004

Bottleneck effects on the sika deer Cervus nippon population in Hokkaido, revealed by ancient DNA analysis.

The population size of the sika deer Cervus nippon on Hokkaido Island of Japan had been remarkably reduced because of heavy hunting pressure since the beginning of Meiji Period and effects of heavy snow in 1879 and 1881. After that, the number of sika deer in Hokkaido has increased gradually due to the protection by the Hokkaido government. In the present study, in order to investigate the bottleneck effects, we analyzed ancient mitochondrial DNA (mtDNA) on sika deer bones excavated from archaeological sites just before Meiji Period. On 86 of 113 bones from 13 archaeological sites of Ainu Culture Period (17-19th centuries), 602 base-pair fragments of the mtDNA control region were successfully sequenced. Consequently, we found three new haplotypes (g-, h- and i-types) which had not been identified in modern sika deer. In addition, four haplotypes (a-, b-, c- and d-types) identified from modern sika deer were also found in the archaeological deer. The new haplotypes and previously reported hapoltypes from sika deer of Hokkaido were phylogenetically much closer to each other, compared with those of modern sika deer from Honshu, Kyushu and the Chinese continent. Geographical distribution patterns of haplotypes of the ancient population were different from those of the modern population in Hokkaido. Our findings indicated that their genetic diversity was reduced through the bottleneck and that population structures of sika deer were changed widely in Hokkaido due to genetic drift.

Animals↗
PubMed1994

Genetic studies of lactococcal bacteriophages--taxonomic differentiations and DNA analysis: evidence for 3' cohesive ends.

Twenty-four bacteriophages of Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were classified. Two groups of bacteriophages morphologically defined as prolate or isometric types by electron microscopy were examined for their genome sizes, protein patterns and DNA homologies. These criteria showed that prolate phages are quite homogeneous. In contrast, isometric phages exhibit more differences, particularly in particle sizes and protein compositions. Analysis of DNA hybridizations confirmed that prolate phages can be grouped together as can be isometric phages but for one exception, phage I52. These two families were clearly defined. The unique phage which does not fit in either group probably belongs to a third one which is much less represented. No obvious relationships between these criteria and the lytic spectra were detected. Evidence of the presence of cohesive ends in phage genomes is also presented in this study. A more detailed analysis performed on one member of the prolate group revealed 3' protruding ends made up of around 13 nucleotides on complementary single strands.

Bacteriophages↗
PubMed1991

Flow cytometric DNA analysis of normal and pathologic parathyroid glands.

Assessment of cellular DNA content and cell cycle analysis by flow cytometry has been useful in predicting patient survival and the malignant potential of many neoplasms. In this study, we analyzed the DNA content and cell cycle distribution of 12 parathyroid adenomas, 15 carcinomas, 5 secondary hyperplasias, and 14 normal parathyroid glands. Appropriate areas demonstrating the pathologic changes were dissociated from paraffin-embedded, formaldehyde-fixed tissue for this purpose. All the cells from normal glands were diploid with mean and median S-phase fractions of 1.2% (range, 0.3-2.7%). Of the 18 glands from five patients with secondary hyperplasia, 4 (22%) had some cells which were aneuploid (from three patients), and 14 (78%) had only cells with a diploid DNA content. Mean and median S-phase fractions were 0.8% (range, 0.3-1.5%). One (8%) of the adenomas was tetraploid, 3 (25%) were aneuploid, and 8 (67%) were diploid. The mean S-phase fraction was 1.5% (range, 0.3-3.8%). Four (27%) of the carcinomas had tetraploid cells, 6 (40%) had aneuploid cells, and 5 (33%) had only diploid cells. Carcinomas had a mean S-phase fraction of 6.0% (median, 5.3%; range, 1.4-14.1%). This study indicates that DNA aneuploidy or tetraploidy may be present in many abnormal parathyroid entities, but not in normal parathyroids. Proliferative activity when considered with the DNA index may be a helpful adjunct in separating carcinomas from adenomas or hyperplasias.

Adenoma↗
PubMed2001

X-linked ocular albinism (Nettleship-Falls): a novel 29-bp deletion in exon 1. Carrier detection by ophthalmic examination and DNA analysis.

BACKGROUND: Mutations in the OA1 gene on the short arm of the X chromosome are known to cause X-linked ocular albinism (x1OA) in males. A four-generation family with this disorder, including asymptomatic carrier females, was investigated by molecular analysis of the OA1 gene. METHODS: DNA samples were available from 22 individuals of this family, including 6 affected males and 6 obligate carriers. The nine exons of the OA1 gene were amplified and further analyzed by SSCP and sequencing. RESULTS: A detailed clinical examination of the index patient and two female carriers showed the typical signs of ocular albinism. Visual evoked potential responses showed markedly asymmetrical responses from the two hemispheres in the affected person as well as in the carriers, as a result of misrouting and decussation of optic nerve fibers. Molecular genetic analysis demonstrated a previously undescribed 29-bp deletion at position 225-253 in exon 1 of the OA1 gene, which segregated in the family. CONCLUSION: Clinical examination combined with molecular genetic analysis enhances the potential for a precise diagnosis for persons at risk of x1OA and provide an accurate basis for genetic counseling.

Adult↗
PubMed1991

[DNA analysis and prenatal diagnosis in cystic fibrosis].

Cystic fibrosis (CF) is the most common autosomal recessive lethal genetic disorder in man, affecting 1 in 2,000 live births. The carrier frequency in Caucasians is 1 in 20. Approximately 70 per cent of the mutations correspond to a specific deletion on chromosome 7 (region 7q31). This delta F 508 mutation of the CF gene results in the loss of phenylalanine at position 508 in the gene product. About 50 per cent of the individuals with CF are homozygous for this deletion and suffer from pancreatic insufficiency. The remaining half are mostly compound heterozygotes between this deletion and another mutation. They exhibit less severe symptoms. Several simple polymerase chain reaction (PCR) tests are available to detect the delta F 508 mutation, allowing rapid prenatal diagnosis of the disease. In those families where other mutations of the CF gene are involved prenatal diagnosis is performed by means of tightly linked restriction fragment length polymorphisms (RFLPs). PCR tests are available for RFLP analysis too. Testing for the delta F 508 deletion, present in about 3 per cent of the normal population, may prove invaluable for extensive carrier screening. Thus, half of the couples at risk could be recognized before the first affected child is born.

Amino Acid Sequence↗