Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “NUCLEASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,207 records · Page 67Linked to original sources
PubMed2000

Using the fluorogenic 5' nuclease assay for high-throughput detection of (CA)n repeats in radiation hybrid mapping.

Here, the power of the 5' nuclease assay to detect PCR products containing (CA)n repeats was compared with that of the classical electrophoretic analysis. This assay, which relies on the use of a unique (CA)10 energy transfer-labeled probe and the 5' nuclease activity of Taq DNA polymerase, was used to construct a dog radiation hybrid map consisting of microsatellite markers. Data from over 7000 PCRs were analyzed in parallel by the fluorogenic assay and the conventional ethidium bromide-stained, agarose gel-based assay. We show that the fluorogenic assay provides a sensitive, reliable and specific method for detecting (CA)n amplimers. Moreover, as no processing is required after the PCR, the risk of carryover contamination and the time required for sample analysis are greatly reduced. All radiation hyrid (RH) assays can be performed using a single PCR protocol, and a standard analysis method has been developed that enables numerically automated data processing. On the whole, using this strategy greatly enhanced the rapidity, throughput and accuracy of the RH mapping of microsatellite markers.

5'-Nucleotidase↗
PubMed2003

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR.

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.

Bacteria↗
PubMed1997

Double-labeled fluorescent probes for 5' nuclease assays: purification and performance evaluation.

An inexpensive method for the purification and evaluation of user-synthesized or crude commercially prepared double-labeled fluorescent probes is presented. These probes exhibit the characteristics required for use in 5'-nuclease assays, including efficient reporter dye quenching, target specificity and susceptibility to cleavage by Taq DNA polymerase during PCR amplification. The method is suitable for research laboratories that wish to develop 5' nuclease assays for the detection of PCR-amplified target sequences to eliminate the requirement for agarose gels and to advance throughput.

5'-Nucleotidase↗
PubMed1984

DNA homology studies in Streptomyces using S1 nuclease.

The optimal reaction conditions for the determination of DNA-homology in Streptomyces species were established in the presence of formamide using S1 nuclease. The melting temperature of Streptomyces DNA was 90 degrees C in 0.42 M NaCl containing 20% formamide in which the denaturation was completed by boiling for 5 minutes. In the S1 reaction mixture consisting of 5 U of S1 nuclease, 0.168 M NaCl, 1 mM ZnSO4 and 8% formamide at pH 4.8, single-stranded DNA was hydrolyzed by more than 98%, while the hydrolysis of double-stranded DNA was less than 3%. From the analysis of homoduplex formation, the C0t1/2 was found at 20 hours, when a mixture of unlabeled DNA and index DNA was used at a ratio of 500:1.

Base Sequence↗
PubMedDate not supplied

[Polydispersity of Serratia marcescens nuclease at optimal pH values].

Treatment with dimethyl suberimidate, a cross-linking bifunctional agent, showed that Sm1 and Sm2 nucleases of Serratia marcescens B10M1 are polydisperse in solution and consist of monomers and dimers at the level of pH optimal for the enzyme activity. The data suggest that nucleases from the strain B10M1 and any other strain are polydisperse at pH optimum if their amino acid sequences are identical.

Amino Acid Sequence↗
PubMed2000

5' Nuclease assays for the loci CCR5-+/Delta32, CCR2-V64I, and SDF1-G801A related to pathogenesis of AIDS.

BACKGROUND: Variations within the human genome play important roles in human disease. To study variations related to susceptibility to AIDS, we have developed 5' nuclease assays that eliminate post-PCR molecular biology steps. METHODS: TaqMan assays based on the 5' nuclease activity of Taq polymerase and fluorescent resonance energy transfer were developed to score alleles at the biallelic loci CCR5-+/Delta32, CCR2-V64I and SDF1-G801A. For each assay, 72 samples were analyzed. Data collection and analysis were performed on the Prism 7700 Sequence Detection System. For comparison with gel electrophoresis methods, each locus was also scored on a subset of 24 samples, using restriction enzymes or single-strand conformational polymorphism (SSCP). RESULTS: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores. CONCLUSIONS: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Time course of serum nuclease activity in mice infected with Plasmodium berghei].

The present paper shows that murine serum nuclease activity increases following P.berghei infection. DNA activity begins increasing just 48 hours after infection. Following 78 hours, it achieves the maximum, by exceeding the baseline level by 6 times. Then DNA activity starts decreasing and following 94 hours after infection it is just thrice higher than the baseline. Serum RNA activity shows only 30% increases 72 hours after infection and returns to the baseline following 94 hours. Microscopic monitoring indicates that only single malaria causative agents appear in the red blood cells in this period. The peak increases in nuclease activities after P.berghei infection are assumed to be associated with the induction of serum infection.

Acute-Phase Proteins↗
PubMed2000

Identification and characterization of nuclease activities in anaerobic environmental samples.

DNA-degrading activity from anaerobic samples of bovine ruminal fluid, primary anaerobic digestor wastewater, freshwater sediments, and marine sediments was observed in the presence of 5 mM EDTA. Nuclease activity experiments involved exposing salmon chromosomal DNA to the environmental samples in 50 mM pH 7.2 buffer, incubating at 37 degrees C, and subjecting the products to electrophoresis. The same stock and concentration of EDTA used in these assays (5 mM) completely inhibited commercial grade DNase. Nuclease activity in two of the samples, ruminal fluid and wastewater, was further characterized. DNA degradation in the ruminal sample was significantly reduced when EDTA or citrate concentrations were increased to 50 mM or above. DNA degradation activity in ruminal fluid was associated with material that passed through a 0.22-micron filter, but wastewater activity was associated with material retained by a 3-micron filter. Degradation activity in the wastewater was resistant to heat pretreatment, whereas the rumen activity was heat-labile (70 degrees C, 60 min). These results demonstrated the biochemical complexity of these two environments and that high molecular weight DNA has a short half-life in these anaerobic environments.

Anaerobiosis↗
PubMed1975

Activity of wheat seedling nuclease toward single-stranded nucleic acids.

Procedures have been developed for the facile preparation of wheat seedling nuclease in highly purified form. The preparation appears to be homogeneous by many physical criteria; however, analytical gel electrophoresis reveals 12 protein bands, only one of which is catalytically active. It is suggested that most of the inactive species are artifacts formed from active enzyme by processes which increase the negative charge of the protein. The enzyme rapidly catalyzes the hydrolysis of denatured DNA and RNA to acid-soluble products and also of the 3'-phosphomonoester linkage of a variety of 3'-mononucleotides. The enzyme has very little, if any, activity toward native DNA with respect to the production of acid-soluble substances; however, athe succeeding paper demonstrates that native DNA is cleaved at a few specific loci to yield large duplex DNA fragments. The nuclease has been characterized as having endonucleolytic activity towards denatured DNA and primarily exonucleolytic activity towards RNA. The mononucleotides produced under the influence of the enzyme bear 5'-phosphomonoester groups. Various lines of evidence indicate a relatively high preference of the enzyme for the hydrolysis primarily of 3'-phosphoester linkages of adenylic acid units and secondarily of thymidylic or uridylic acid units in DNA and in ribohomopolymers, respectively. Corresponding linkages involving cytidylic acid and especially guanylic acid are relatively resistant. The 3'-nucleotidase activity of the enzyme at pH 5.0 towards the various mononucleotides fits the same pattern; i.e. nucleotides containing adenine are hydrolyzed most rapidly, followed in decreasing order by those containing thymine or uracil, cytosine, and guanine.

Centrifugation, Density Gradient↗
PubMed2002

Nucleic acid molecular biomarkers for diagnostic biodosimetry applications: use of the fluorogenic 5'-nuclease polymerase chain reaction assay.

A reliable, relatively easy method for diagnostic assessment of radiation exposure is needed to support the triage of radiation casualties and medical treatment decisions in military defense operations. Our strategy is to identify radiation-responsive DNA mutations and gene expression targets that can be analyzed using polymerase chain reaction (PCR) assays and an existing fluorescence-based nucleic acid analysis system designed for forward-deployable laboratory applications. Using an in vitro model system of human peripheral blood lymphocytes, we identified a candidate nucleic acid biomarker (i.e., gene expression target) that is responsive to ionizing radiation. In this report, we describe our preliminary Haras gene expression findings. A dose-dependent elevation in Haras gene expression levels was demonstrated using Northern-blot analysis 17 hours after exposure to a 250-kVp dose of X-rays (25-100 cGy, 1 Gy/minute); c-Haras expression levels at 100 cGy were ninefold higher than those of controls. An alternative protocol to quantify the Haras cDNA target, using the rapid, real-time reverse transcriptase fluorogenic 5'-nuclease PCR assay, is described, along with a preliminary characterization of the dynamic range for detection. Our research shows that the analysis of multitarget nucleic acid biomarkers, using the multiplex fluorogenic 5'-nuclease PCR assay, has beneficial applications in radiation epidemiology, radiation therapy, and biodosimetry.

Humans↗
PubMed2003

[Coding single nucleotide polymorphism is an ideal marker for detecting gene imprinting by 5' nuclease assay].

OBJECTIVE: To establish a novel approach for quick and high throughput verification of human gene imprinting. METHODS: By use of a pair of dye-labeled probes, 5' nuclease assay was combined with reverse transcriptase-PCR(RT-PCR) to genotype a coding single nucleotide polymorphism (cSNP), rs705(C/T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lymphoblast cell lines. RESULTS: Allele discrimination showed a clear monoallelic expression pattern of SNRPN, which was confirmed by RT-PCR based restriction fragment length polymorphisms. Pedigree analysis verified the paternal origin of expressed allele, which is in consistency with previous report. CONCLUSION: Coding SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach has also a potentiality to discover differential allele expression of non-imprinted genes in order to find gene cis-acting functional polymorphism.

Alleles↗
PubMed2001

[The nucleic acid analysis by DNA chip technique based on nuclease S1 protection].

OBJECTIVE: Establishing a method for quantitative analysis of nucleic acid by DNA chips. METHODS: The modified oligonucleotides with ribose at 3'-end was chemically synthesized. The 5'-end was labeled by radioisotope 32P with kinase catalyzed reactions. Such oligonucleotides were converted into di-aldehyde at 3'-end by oxidization with NaIO4, and then were spotted on glass slide with the amino group modified surface. After reduced with NaBH4, the oligonucleotides were attached strongly. The DNA chips prepared with this method were hybridized with nucleic acids existed in the solution and then digested with nuclease S1. RESULTS: When they were paired with the nucleic acids in the solution perfectly, the oligonucleotides on the chip were not cleaved by nuclease S1. Otherwise, the oligonucleotides on chip were cleaved. The protection efficiencies appeared proportional to the perfect paired nucleic acids in the solution when the content of target nucleic acids were less than the spots on the slides. CONCLUSIONS: The method was developed for both qualitative and quantitative analysis of nucleic acid. As it was not required to label the samples with radioisotope or fluorescence, it might be a practical choice for clinical tests.

DNA Probes↗
PubMed2000

[Study on the interaction between nuclease P1 and CuCl2 (II) by visible absorption spectra].

In this paper, we studied the directly interaction between nuclease Pl and CuCl2 in aqueous solution by visible absorption spectra. As a result, the zinc ion in the active site of nuclease Pl can be replaced by additional Cu(II). At the the same time, Cu(II) entered into the active site of enzyme forming the correspondent derivatives of "Cu(II)-Pl". The variety of pH value influenced the types of derivatives and the enzyme activity.

Catalysis↗
PubMed1992

[The isolation of nucleases Sm1 and Sm2 in preparative amounts and a study of their antigenicity].

Two enzyme forms of endonuclease (Sm 1 and Sm 2) strain B10M1 in 60 and 100 mg respectively have been isolated from the culture fluid Serratia marcescens. The chromatographic and electrophoretic properties and N-terminal amino acid residues are different for both enzymes. The purification procedure consists of dialysis and ion-exchange chromatography on DEAE- and phosphocellulose. The yield of nucleases Sm1 and Sm2 are 14% and 28% respectively. The antigenic differences of nucleases Sm1 and Sm2 have been found by cross immunoenzyme analysis.

Animals↗
PubMedDate not supplied

[Immunogenic proteins of nuclease-sensitive regions of chromatin of the normal rat liver and N-nitrosodiethylamine-induced hepatoma].

The localization of immunogenic proteins of chromatin in the nuclease-hypersensitive regions was investigated by means of polyclonal antibodies against the total chromatin from cells N-nitrosodiethylamine-induced hepatoma of rats and from hepatocytes. The fractions released after digestion by Ca-dependent endonuclease and by exogenous DNasa-1 were considerably enriched with immunogenic determinants. The enrichment of nuclease-hypersensitive sites of chromatin with tumour-associated antigens was observed in experiments with antihepatoma antibodies preincubated with chromatin from the normal liver cells. Higher immunoreactivity was typical of fractions obtained during Ca-dependent endonuclease segregation of the hepatoma chromatin, while products of digestion by exogenous DNasa-1 possessed higher tumour specificity.

Animals↗
PubMed1992

Action of a nuclease from rat nuclei on UV-irradiated DNA.

An Mg(2+)-dependent nuclease was highly purified from rat-liver nuclei. The nuclease activity was enhanced in ultraviolet light (UV)-irradiated dsDNA, but not in unirradiated dsDNA. Irrespective of UV irradiation, ssDNA was readily cleaved by this enzyme. UV-irradiated plasmid DNA was incubated with this enzyme and subjected to the template primer activity assay with a Klenow polymerase. With increasing incubation time, the activity was enhanced in the circular-relaxed and linear forms, but not in the superhelical form. These results implied that this enzyme excises UV-damaged sites in dsDNA to form single strand gaps for repair synthesis.

Animals↗
PubMed1977

[Study of rat brain tissue nucleases].

Acid and alkaline nucleases of the brain tissues have been identified and partially purified. Alkaline DNA-ase hydrolyzing denatured DNA at pH 8,0; alkaline RNA-ase having optimal activity at pH 8,0 and nuclease intensively hydrolyzing both DNA and RNA at pH 5.0. The molecular weights of these enzymes have been determined.

Animals↗