Synchrony of Digestion of SV40 DNA by Exonuclease III Determined by EM

1975 ◽  
Vol 33 ◽  
pp. 674-675
Author(s):  
Ray Wu ◽  
G. Ruben ◽  
B. Siegel ◽  
P. Spielman ◽  
E. Jay
Keyword(s):  
Dark Field ◽  
Uranyl Acetate ◽  
Enzyme Digestion ◽  
Dna Molecules ◽  
Exonuclease Iii ◽  
Aluminum Films ◽  
Double Stranded Dna ◽  
Before And After ◽  
Exo Iii ◽  
Sv40 Dna

A method for determining long nucleotide sequences of double-stranded DNA is being developed. It involves (a) the synchronous digestion of the DNA from the 3' ends with EL coli exonuclease III (Exo III) followed by (b) resynthesis with labeled nucleotides and DNA polymerase. A crucial factor in the success of this method is the degree to which the enzyme digestion proceeds synchronously under proper conditions of incubation (step a). Dark field EM is used to obtain accurate measurements on the lengths and distribution of the DNA molecules before and after digestion with Exo III, while gel electrophoresis is used in parallel to obtain a mean length for these molecules. It is the measurements on a large enough sample of individual molecules by EM that provides the information on how synchronously the digestion proceeds. For length measurements, the DNA molecules were picked up on 20-30 Å thick carbon-aluminum films, using the aqueous Kleinschmidt technique and stained with 7.5 x 10-5M uranyl acetate in 90% ethanol for 3 minutes.

Improved Resolution of Stained SV40 DNA Fragments in Dark Field EM

1975 ◽  
Vol 33 ◽  
pp. 660-661
Author(s):  
G. Ruben ◽  
E. Jay ◽  
R. Wu ◽  
B. Siegel
Keyword(s):  
Cytochrome C ◽  
Basic Protein ◽  
Restriction Enzymes ◽  
Dark Field ◽  
Dna Fragments ◽  
Bright Field ◽  
Field Technique ◽  
Double Stranded Dna ◽  
Suitable Restriction ◽  
Sv40 Dna

The technique for spreading double and single-stranded DNA using a basic protein developed by Kleinschmidt is routine and widely used. After shadowing DNA prepared by this method the cytochrome c on the DNA and in the background gives an apparent increase in length and width of the DNA. The smallest DNA length measurement which has been reported for homogeneous double-stranded molecules is 1300 Å using the Kleinschmidt technique in bright field. In bright field, the uneven background of the cytochrome c film and metal deposit makes size measurements of shorter DNA fragments difficult. In the dark field technique, the resolution limitation is imposed by the stained width of the DNA itself. Thus in principle, much shorter lengths should be measurable.The resolution limits on double-stranded DNA by the dark field technique have been tested by using suitable restriction enzymes to cleave SV40 DNA to produce a number of discrete DNA fragments of defined lengths.

Ultrasensitive fluorometric biosensor based on Ti3C2 MXenes with Hg2+-triggered exonuclease III-assisted recycling amplification

The Analyst ◽  
2021 ◽  
Author(s):  
Liling Lu ◽  
Xiao Han ◽  
Jingwen Lin ◽  
Yingxin Zhang ◽  
Minghao Qiu ◽  
...  
Keyword(s):  
Two Dimensional ◽  
Exonuclease Iii ◽  
Exo Iii ◽  
Recycling Amplification ◽  
Fluorescence Quencher

Herein a rapid and sensitive fluorometric bioanalysis platform for mercury(II) (Hg2+) detection was innovatively developed using ultrathin two-dimensional MXenes (Ti3C2) as fluorescence quencher and Hg2+-induced exonuclease III (Exo III)-assisted target...

scholarly journals Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions

2020 ◽  
pp. 153537022097397
Author(s):  
Maria Troisi ◽  
Mitchell Klein ◽  
Andrew C Smith ◽  
Gaston Moorhead ◽  
Yonatan Kebede ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Therapeutic Potential ◽  
Dnase I ◽  
Protein Recognition ◽  
Exonuclease Iii ◽  
Branched Dna ◽  
Acid Cleavage ◽  
Nuclease Digestion ◽  
Nuclease Resistance ◽  
Exo Iii

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions. Hairpins are inserted to reduce end fraying and effectively eliminate potential nuclease binding sites. We compare the structure and protein recognition features of J1 with four intramolecular four-way junctions: i-J1, i-J1(PS1), i-J1(PS2) and i-J1(PS3). Circular dichroism studies suggest that the secondary structure of each intramolecular 4WJ is composed predominantly of B-form helices. Thermal unfolding studies indicate that intramolecular four-way junctions are significantly more stable than J1. The Tm values of the hairpin four-way junctions are 25.2° to 32.2°C higher than the control, J1. With respect to protein recognition, gel shift assays reveal that the DNA-binding proteins HMGBb1 and HMGB1 bind the hairpin four-way junctions with affinity levels similar to control, J1. To evaluate nuclease resistance, four-way junctions are incubated with DNase I, exonuclease III (Exo III) and T5 exonuclease (T5 Exo). The enzymes probe nucleic acid cleavage that occurs non-specifically (DNase I) and in a 5ʹ→3ʹ (T5 Exo) and 3ʹ→5ʹ direction (Exo III). The nuclease digestion assays clearly show that the intramolecular four-way junctions possess significantly higher nuclease resistance than the control, J1.

Label-free DNA detection based on oligonucleotide-stabilized silver nanoclusters and exonuclease III-catalyzed target recycling amplification

2014 ◽  
Vol 6 (15) ◽  
pp. 6082-6087 ◽  
Author(s):  
Hui Ma ◽  
Wei Wei ◽  
Qian Lu ◽  
Zhixin Zhou ◽  
Henan Li ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Silver Nanoclusters ◽  
Label Free ◽  
Exonuclease Iii ◽  
Target Recycling ◽  
Free Dna ◽  
Sensitivity And Selectivity ◽  
Exo Iii ◽  
Ag Ncs ◽  
Recycling Amplification

A label-free DNA biosensor with high sensitivity and selectivity is constructed by using DNA–Ag NCs and Exo III-catalyzed target recycling amplification.

DNA topoisomerase activity associated with simian virus 40 nucleoprotein complexes

1994 ◽  
Vol 72 (5-6) ◽  
pp. 195-201 ◽  
Author(s):  
Claude Hamelin ◽  
Benoit D'Amours ◽  
Christian Page ◽  
Young Sup Chung
Keyword(s):  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Topoisomerase ◽  
Infected Monkey ◽  
Before And After ◽  
Catalytically Active ◽  
Nucleoprotein Complexes ◽  
Sv40 Dna

Simian virus 40 (SV40) chromatin extracted from nuclei of infected monkey cells (CV1) was sedimented in neutral sucrose gradients, before and after digestion with bovine pancreatic RNase I-A or DNase I. DNA topoisomerase (TI) activity was found associated with RNase-resistant, DNase-sensitive SV40 nucleoprotein complexes. After polyacrylamide gel electrophoresis, a number of proteins with a molecular mass between 40 and 70 kDa were seen at the level of viral DNA peaks, some of which may represent catalytically active breakdown products of the TI enzyme. Large protein complexes were observed under the electron microscope in association with the viral chromosomes and appear to correspond to the SV40 DNA replication complex, including TI. Our results suggest that TI activity is indeed associated with the viral minichromosomes undergoing replication in vivo.Key words: deoxyribonucleoproteins, DNA topoisomerase, minichromosomes, ribonucleoproteins, simian virus 40, viral chromatin.

scholarly journals Changes in the Level of DNA Fragmentation in Sperm Cells detected by Acridine Orange Test in Men with Sub/infertility Treated with Nutritional Supplement PAPA

Folia Medica ◽  
2020 ◽  
Vol 62 (1) ◽  
pp. 112-116
Author(s):  
Stoil N. Tomov ◽  
Radoslava St. Stoyanova ◽  
Pepa K. Atanasova ◽  
Ivan Y. Dechev
Keyword(s):  
Acridine Orange ◽  
Dna Fragmentation ◽  
Physiological Condition ◽  
Nutritional Supplement ◽  
Sperm Cells ◽  
Sperm Dna Fragmentation ◽  
Double Stranded Dna ◽  
Sperm Dna ◽  
Before And After ◽  
Sperm Nuclei

Background: When diagnosing and treating male infertility it is important to determine whether there are defects in the maturation process of sperm nuclei. Using nutritional supplements can improve the morphological and physiological condition of the spermatozoa. In recent years there has been an increase in the usage of supplements with different compositions which strives to determine the best combination and avoid side effects. &nbsp; Aim: To study the effect of PAPA nutritional supplement on the levels of DNA fragmentation of sperm cells tested with acridine orange test (single stranded DNA against double stranded DNA) in men with sub/infertility. &nbsp; Materials and methods: 48 men with confirmed sub/infertility underwent treatment for three months with nutritional supplement PAPA containing 9 micronutrients. The differences in levels of DNA fragmentation were determined with acridine orange test, which was conducted before and after the treatment. &nbsp; Results: The results were statistically significant (p<0.001) showing an increase in the number of green spermatozoa (normal DNA), and a decrease of damaged ones (orange and red). After treatment the level of sperm DNA fragmentation decreased by 10.2%. &nbsp; Conclusion: Men with confirmed sub/infertility that took nutritional supplement PAPA for three moths showed a decrease in DNA fragmentation levels of 10.2% determined by AO test which implies an improvement of male fertility levels.

Target-activated cascaded digestion amplification of exonuclease III aided signal-on and ultrasensitive fluorescence detection of ATP

2018 ◽  
Vol 42 (5) ◽  
pp. 3534-3540 ◽  
Author(s):  
Xueqi Leng ◽  
Rongguo Li ◽  
Yu Wang ◽  
Yunping Wu ◽  
Yuqin Tu ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Fluorescence Detection ◽  
Fluorescence Sensing ◽  
Exonuclease Iii ◽  
One Step ◽  
Exo Iii

In this work, a rapid, one-step and ultrasensitive signal-on fluorescence sensing for the detection of adenosine triphosphate (ATP) based on target-activated cascaded digestion amplification with Exo III aid was developed.

scholarly journals Turn-On Fluorescence Aptasensor on Magnetic Nanobeads for Aflatoxin M1 Detection Based on an Exonuclease III-Assisted Signal Amplification Strategy

Nanomaterials ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 104
Author(s):  
Fuyuan Zhang ◽  
Linyang Liu ◽  
Shengnan Ni ◽  
Jiankang Deng ◽  
Guo-Jun Liu ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Aflatoxin M1 ◽  
Exonuclease Iii ◽  
Sensor Performance ◽  
Milk Samples ◽  
Increased Sensitivity ◽  
Exo Iii ◽  
Amplification Strategy ◽  
Optimized Conditions ◽  
Magnetic Nanobeads

In order to satisfy the need for sensitive detection of Aflatoxin M1 (AFM1), we constructed a simple and signal-on fluorescence aptasensor based on an autocatalytic Exonuclease III (Exo III)-assisted signal amplification strategy. In this sensor, the DNA hybridization on magnetic nanobeads could be triggered by the target AFM1, resulting in the release of a single-stranded DNA to induce an Exo III-assisted signal amplification, in which numerous G-quadruplex structures would be produced and then associated with the fluorescent dye to generate significantly amplified fluorescence signals resulting in the increased sensitivity. Under the optimized conditions, this aptasensor was able to detect AFM1 with a practical detection limit of 9.73 ng kg−1 in milk samples. Furthermore, the prepared sensor was successfully used for detection of AFM1 in the commercially available milk samples with the recovery percentages ranging from 80.13% to 108.67%. Also, the sensor performance was evaluated by the commercial immunoassay kit with satisfactory results.

Sign in / Sign up

Export Citation Format

Share Document