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At least 667 records · Page 37Linked to original sources
PubMed2002

Chelating resin-based extraction of DNA from dental pulp and sex determination from incinerated teeth with Y-chromosomal alphoid repeat and short tandem repeats.

A procedure utilizing Chelex 100, chelating resin, was adapted to extract DNA from dental pulp. The procedure was simple and rapid, involved no organic solvents, and did not require multiple tube transfers. The extraction of DNA from dental pulp using this method was as efficient, or more so, than using proteinase K and phenol-chloroform extraction. In this study, the Chelex method was used with amplification and typing at Y-chromosomal loci to determine the effects of temperature on the sex determination of the teeth. The extracted teeth were incinerated in a dental furnace for 2 minutes at 100 degrees C, 200 degrees C, 300 degrees C, 400 degrees C, and 500 degrees C. After the isolation of DNA from the dental pulp by the Chelex method, alphoid repeats, and short tandem repeats, the human Y chromosome (DYZ3), DYS19, SYS389, DYS390, and DYS393 could be amplified and typed in all samples incinerated at up to 300 degrees C for 2 minutes. The DYS389 locus in some samples could not be amplified at 300 degrees C for 2 minutes. An autopsy case is described in which genotypings of DYS19, DYS390, and DYS393 from dental pulp obtained from a burned body were needed. The data presented in this report suggest that Chelex 100-based DNA extraction, amplification, and typing are possible in burned teeth in forensic autopsy cases.

Chelating Agents↗
PubMed2001

Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples.

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers amplifying C. neoformans URA5 gene sequences. To test compatibility of different DNA extraction protocols with the PCR-restriction fragment length polymorphism (RFLP) assay, a commercial DNA extraction kit (XTRAX; Gull Laboratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted DNA from spiked clinical samples containing C. neoformans (8 +/- 2 cells ml(-1)) and generated amplification products suitable for restriction enzyme analysis. Alu I digestion differentiated the two varieties of C. neoformans. Three distinct RFLP patterns were obtained upon restriction with MspI corresponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analysis, coupled with a sensitive in-house or commercially available DNA extraction method from clinical samples, could be successfully incorporated into rapid routine diagnostic strategies. It could also provide an expeditious tool for epidemiology-based population genetics studies.

Base Sequence↗
PubMed2001

Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA.

AIM: To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. METHODS: DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the beta globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. RESULTS: Microwave extraction showed the highest positive rate for beta globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp beta globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 beta globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the beta globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. CONCLUSIONS: HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered.

Carcinoma, Squamous Cell↗
PubMed2004

Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex.

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.

Cell-Free System↗
PubMed1989

Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms.

Two methods were compared for the extraction of DNA from small numbers of bacterial cells. The first method involved lysis of cells with SDS in the presence of proteinase K, treatment with hexadecyltrimethyl ammonium bromide (CTAB) and precipitation of DNA with isopropanol. In the second method, DNA was extracted by treatment of the cells with guanidine hydrochloride (GHCl) and precipitated with ethanol. Thirty strains of representative gram positive and gram negative species were included in the study. Preparations derived from confluent growth on one-quarter of the surface of agar plates and from 10(8) cells were subjected to each extraction procedure and analyzed for their content of DNA, RNA and protein. The suitabilities of the resultant DNA for restriction enzyme digestion and biotin-labelling by a random primer technique were also assessed. In general, the CTAB method yielded greater amounts of DNA than the GHCl procedure. RNA was present in most preparations of both types, but in amounts detectable only by agarose gel electrophoresis. The latter technique also revealed that DNA was not excessively sheared by either procedure. Protein was detected in some CTAB and GHCl preparations, but was not consistently associated with one or the other method. DNA obtained by both methods could be digested by the restriction enzyme EcoR I. In addition, biotin-labelled DNA probes prepared from CTAB and GHCl preparations were capable of hybridizing with homologous target DNA fixed to nitrocellulose. Since the CTAB method was consistently successful in recovering DNA from preparations containing 10(8) cells, it may be more suitable for the direct treatment of single colonies taken from primary isolation plates or plaque samples.

Bacteria↗
PubMed1992

Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin.

A new method for the extraction of bacterial DNA from soil has been developed. Soil samples of 50 g were dispersed, and bacteria were released by use of a cation-exchange resin; subsequently, bacteria were separated from soil particles by low-speed centrifugation and lysed with lysozyme and ionic detergent, and the DNA was then purified by CsCl-ethidium bromide equilibrium density centrifugation. The extracted DNA was of high molecular weight and sufficiently pure for restriction enzyme digestion, DNA-DNA hybridization, and amplification by the polymerase chain reaction. The advantages of the new method are that the separation of bacteria from soil is considerably faster than by repeated blending, more samples can be handled, and furthermore no aerosols are formed during separation. Also, we investigated whether the CsCl-ethidium bromide equilibrium density centrifugation could be replaced by purification using Gene-Clean. However, this method produced DNAs which were insufficiently pure for several types of analysis. The new method was used to study survival of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading Pseudomonas cepacia DBO1 (pRO101) in unamended soil and in soil amended with 2,4-D. We found that the degrading strain, irrespective of inoculation level, was able to grow to the same high numbers in soil amended with 2,4-D, while the strain in nonamended soil were maintained at the inoculation level. Detection based on DNA extraction and subsequent dot blot DNA-DNA hybridization was in accordance with detection by plating on selective medium.

Journal Article↗
PubMed1987

Extraction of cellular DNA from human cells and tissues fixed in ethanol.

DNA can be extracted from ethanol-fixed lymphoid cells and tissues. The fixation procedure is simple and rapid, and the DNA extraction itself is the same as that normally used for fresh tissue or cells. DNA extracted from ethanol-fixed material is indistinguishable from DNA extracted from fresh samples based on its purity, its ability to be digested with restriction endonucleases, and its ability to specifically hybridize to DNA probes. The capability to extract DNA from ethanol-fixed cells and tissues eliminates the need for stringent handling and storage requirements of fresh or frozen specimens.

DNA↗
PubMed1995

A rapid method for extraction and purification of DNA from dental plaque.

A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.

Base Sequence↗
PubMed2005

High-throughput molecular testing of ticks using a liquid-handling robot.

To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 +/- 0.04 microg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-microl volumes with a reproducibility of +/- 0.038 microl when dispensing 1.0 microl. By using this process, 96 samples can be extracted and tested while reducing human labor to approximately 30 min.

Animals↗
PubMed2003

Extraction of Chlamydia pneumoniae DNA from vascular tissue for use in PCR: an evaluation of four procedures.

The objective of this study was to compare four procedures for Chlamydia pneumoniae DNA extraction from vascular tissue. The NucliSens Kit, the QIAamp tissue DNA MiniKit, buffer-saturated phenol and the Geneclean II Kit were evaluated, based on the yield of recovered DNA, using PCR to detect C. pneumoniae in vascular tissue. The QIAamp tissue procedure had the highest detection level (0.004 inclusion-forming units/sample). All methods, except NucliSens (70 min), had a short handling time (30-40 min). Costs varied from 0.5 to 3.2 Euro.

Chlamydia Infections↗
PubMed1995

[Fundamental and practical study for DNA analysis using tooth as a source of DNA].

Degree of degradation and the yield of DNA extracted from dental pulp tissues were examined on the tooth samples (n = 50) stored at room temperature and the method of DNA extraction from tooth hard tissues was also investigated. The DNA samples obtained were also applied to forensic odontological material examination including DNA fingerprinting using a probe Myo and VNTR (variable number of tandem repeat) analysis in D4S43 locus by PCR. The amount of DNA obtained from the dental pulp tissue of a single tooth varied approximately from 3 to 40 micrograms. In most cases, high molecular weight DNA was still present in samples stored at room temperature for at least 336 days. When the dental pulp tissue samples were less than 5 mg in weight, the amount DNA extracted was usually less than 10 micrograms, however when the samples were more than 5 mg in weight, the amount of DNA extracted was more than 10 micrograms. No correlation was observed between the storage period of the tooth samples and the DNA extraction ratio (the amount of extracted DNA weight, micrograms/pulp weight, mg). The efficiency of DNA extraction from tooth hard tissues was investigated under different conditions using 0.005 M and 0.5 M EDTA solutions for decalcification. DNA was efficiently extracted from the tooth samples which were decalcified for one week without changing the 0.5 M EDTA solution or by changing the solution once within a week. Rapid decalcification using formic acid buffer was not suitable for DNA extraction from tooth hard tissues. Southern blot hybridization of DNA samples extracted from pulp tissues using Myo probe gave multiple bands. Finger print patterns obtained from DNA recovered from dental pulp and tooth hard tissues samples were identical, however, the number of hybridizing bands obtained from tooth hard tissues was less than that obtained from blood and dental pulp tissues. The D4S43 typing using DNA recovered from blood stains, dental pulp tissues and tooth hard tissues of the same individuals was in agreement with each other and the 184bp fragment was efficiently amplified in all the samples tested. The DNA obtained from dental pulp tissues usually contains high molecular weight DNA and was suitable for multilocus probe and PCR analysis. However, the DNA obtained from tooth hard tissues was suitable only for PCR analysis.

Adolescent↗
PubMed2000

DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts.

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

Animals↗
PubMed1976

Factors influencing the yield of satellite DNA in extractions from Drosophila virilis and Drosophila melanogaster adults and embryos.

The application of different DNA extraction methods to identical batches of Drosophila virilis and Drosophila melanogaster flies or embryos has revealed that the ionic strength of a homogenization medium is of critical importance if chloroform extractions are performed. The low yield of satellite DNA after homogenization in low salt buffers is less severe if EDTA is included in the buffer. Phenol extraction procedures result in no such differential behavior of satellite and main band DNA, but under certain circumstances a particular satellite fraction of Drosophila virilis DNA may be lost.

Animals↗
PubMed2005

Quantitative determination of Roundup Ready soybean (Glycine max) extracted from highly processed flour.

Roundup Ready soybean powder has been subjected to different amounts of DNA fragmentation to assess the accuracy of real-time PCR on processed food. Certified reference material (CRM) containing 10 g kg(-1) of Roundup Ready soybean (ERM-BF410d) prepared by a dry-mixing processing method was exposed to water at two temperatures, using three different mixing devices, or to baking temperature (250 degrees C) for 30 min. The amount of DNA extracted from the different samples was quantified by fluorimetry. The amount of fragmentation of the extracted DNA was characterised by gel and capillary electrophoresis and the percentage of genetically modified (GM) soybean was determined by a double quantitative real-time PCR method. Measurement of the event GTS 40-3-2 (RUR) was possible in all the treated materials, because small amplicons were amplified. Correct RUR percentages could be measured for intact powders with little or no DNA fragmentation. For samples with a high level of DNA degradation, however, the accuracy of the measurement was found to depend on the method used for DNA extraction. Genomic DNA isolated by use of silica resin resulted in statistically significant overestimation of the amount of GM.

DNA↗
PubMed1985

A coupled in vitro system for the formation and packaging of concatemeric phage T1 DNA.

Extracts derived from E. coli cells infected non-permissively with phage T1 amber mutants were used in an in vitro system to investigate the packaging of T1 DNA into phage heads. The standard extract used infections with amber mutants in genes 1 and 2 (g1- g2-) which are defective in T1 DNA synthesis but can synthesis the proteins required for particle morphogenesis. g1- g2- extracts packaged T1+ virion DNA molecules with an efficiency of 3 X 10(5) pfu/micrograms DNA. Extracts from cells infected with phage also defective in DNA synthesis but carrying additional mutations in genes 3.5 or 4 which are required for concatemer formation in vivo (g1- g3.5- and g1- g4- extracts) package T1 virion DNA at substantially lower efficiencies. Analysis of the DNA products from these in vitro reaction showed that concatemeric DNA is formed very efficiently by g1- g2- extracts but not by g1- g3.5- or g1- g4- extracts. These results are interpreted as evidence that the T1 in vitro DNA packaging system primarily operates in a similar manner to the in vivo headful mechanism. This is achieved in vitro by the highly efficient conversion of T1 virion DNA into concatemers which are then packaged with a much lower efficiency into heads to form infectious particles. A secondary pathway for packaging T1 DNA into heads and unrelated to the headful mechanism may also exist.

DNA Replication↗
PubMedDate not supplied

Inhibitors present in blood do not inhibit PCR from buccal cell preparations: case report.

BACKGROUND: Hemochromatosis is a common disease that is characterized by high ferritin levels and/or high iron saturation and mutations in two alleles. MATERIAL AND METHODS: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) is often performed on DNA extracted from blood since blood yields high concentrations of DNA. However, inhibitors can cause PCR failure in DNA extracted from blood thus preventing a molecular diagnosis. RESULTS: This report describes a case where multiple blood draws resulted in unamplifiable DNA. Subsequently, a buccal cell sample was collected and extracted. DNA extracted from the buccal cells yielded amplifiable DNA in contrast to DNA extracted from the patient's blood. In addition, the patient was identified as having a homozygous mutation for one allele of the hemochromatosis gene. CONCLUSION: These results suggests that a buccal cell DNA extraction may be useful in cases where blood samples contain inhibitory substances for PCR.

Blood Proteins↗
PubMed1995

In vitro proteolysis of endonucleases in rat liver nuclei extracts.

DNA endonucleases in rat liver nuclei extracts were examined by SDS-polyacrylamide gel electrophoresis followed by zymogram analysis. Four polypeptides of 120, 54, 31 and 28 kDa, which have DNA endonuclease activity, were shown to occur in the extract isolated in the presence of phenylmethanesulfonyl fluoride (PMSF), a proteinase inhibitor. Isolation without PMSF, as well as storage at -20 degrees C, or autodigestion, resulted in multiplication of active polypeptides in the extracts. Trypsin digestion led to the appearance of an active > 140 kDa polypeptide, indicating the existence of a potential endonuclease precursor in the nuclear extract.

Animals↗