scholarly journals Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion

2020 ◽  
Author(s):  
Juan Canoura ◽  
Haixiang Yu ◽  
Obtin Alkhamis ◽  
Daniel Roncancio ◽  
Rifat Farhana ◽  
...  
Keyword(s):  
Exonuclease Digestion

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

scholarly journals Analysis of the psoralen-crosslinking pattern in chromatin DNA by exonuclease digestion

1988 ◽  
Vol 16 (14) ◽  
pp. 7013-7024 ◽  
Author(s):  
R.M. Widmer ◽  
Th. Koller ◽  
J.M. Sogo
Keyword(s):  
Exonuclease Digestion

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

2021 ◽  
Author(s):  
Vandana Kuttappan Nair ◽  
Chandrika Sharma ◽  
Mrittika Sengupta ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Circular Dna ◽  
Rolling Circle ◽  
Specific Amplification ◽  
Exonuclease Digestion ◽  
Sticky End

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

scholarly journals Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (3) ◽  
pp. 1736-1745 ◽  
Author(s):  
Palaniyandi Manivasakam ◽  
Robert H. Schiestl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Enzyme ◽  
Restriction Enzymes ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Dna Integration ◽  
Exonuclease Digestion

ABSTRACT The BamHI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R. H. Schiestl and T. D. Petes, Proc. Natl. Acad. Sci. USA 88:7585–7589, 1991). The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends. Our results show that in addition to BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI,SalI, SmaI, HpaI, MscI, and SnaBI do not. Secondly, the three active enzymes stimulated integrations only of fragments containing 5′ or 3′ overhangs but not of blunt-ended fragments. Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology. Furthermore, an Asp718 fragment possessing a 5′ overhang integrated into a KpnI (isoschizomer) site possessing a 3′ overhang, most likely by filling of the 5′ overhang followed by 5′ exonuclease digestion to produce a 3′ end. We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions. The majority of events integrated into single sites. In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations.

scholarly journals Exonuclease digestion of chromosomes forin situhybridization

1988 ◽  
Vol 16 (21) ◽  
pp. 10381-10381 ◽  
Author(s):  
E. R. Schmidt
Keyword(s):  
Exonuclease Digestion

scholarly journals Processing of the Precursors to Small Nucleolar RNAs and rRNAs Requires Common Components

1998 ◽  
Vol 18 (3) ◽  
pp. 1181-1189 ◽  
Author(s):  
Elisabeth Petfalski ◽  
Thomas Dandekar ◽  
Yves Henry ◽  
David Tollervey
Keyword(s):  
Small Nucleolar Rnas ◽  
Rrna Processing ◽  
5.8S Rrna ◽  
Genes Encoding ◽  
Sequences Analysis ◽  
Upstream Processing ◽  
Exonuclease Digestion ◽  
Nucleolar Rna ◽  
Nucleolar Rnas ◽  
Common Components

ABSTRACT The genes encoding the small nucleolar RNA (snoRNA) species snR190 and U14 are located close together in the genome of Saccharomyces cerevisiae. Here we report that these two snoRNAs are synthesized by processing of a larger common transcript. In strains mutant for two 5′→3′ exonucleases, Xrn1p and Rat1p, families of 5′-extended forms of snR190 and U14 accumulate; these have 5′ extensions of up to 42 and 55 nucleotides, respectively. We conclude that the 5′ ends of both snR190 and U14 are generated by exonuclease digestion from upstream processing sites. In contrast to snR190 and U14, the snoRNAs U18 and U24 are excised from the introns of pre-mRNAs which encode proteins in their exonic sequences. Analysis of RNA extracted from a dbr1-Δ strain, which lacks intron lariat-debranching activity, shows that U24 can be synthesized only from the debranched lariat. In contrast, a substantial level of U18 can be synthesized in the absence of debranching activity. The 5′ ends of these snoRNAs are also generated by Xrn1p and Rat1p. The same exonucleases are responsible for the degradation of several excised fragments of the pre-rRNA spacer regions, in addition to generating the 5′ end of the 5.8S rRNA. Processing of the pre-rRNA and both intronic and polycistronic snoRNAs therefore involves common components.

scholarly journals HIV-1 Preintegration Complexes Preferentially Integrate into Longer Target DNA Molecules in Solution as Detected by a Sensitive, Polymerase Chain Reaction-based Integration Assay

2001 ◽  
Vol 276 (50) ◽  
pp. 46946-46952 ◽  
Author(s):  
Alexei Brooun ◽  
Douglas D. Richman ◽  
Richard S. Kornbluth
Keyword(s):  
Polymerase Chain Reaction ◽  
Host Cell ◽  
Polymerase Chain Reaction Amplification ◽  
Chain Reaction ◽  
Preintegration Complex ◽  
Target Dna ◽  
Host Cell Factors ◽  
A Cell ◽  
Polymerase Chain ◽  
Exonuclease Digestion

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using λ exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

A linear plasmid in Tilletia controversa, a fungal pathogen of wheat

1988 ◽  
Vol 66 (6) ◽  
pp. 1098-1100 ◽  
Author(s):  
W. K. Kim ◽  
S. A. McNabb ◽  
G. R. Klassen
Keyword(s):  
Fungal Pathogen ◽  
Physical Map ◽  
Restriction Enzymes ◽  
Ribosomal Genes ◽  
Puccinia Graminis ◽  
Ustilago Hordei ◽  
Tilletia Controversa ◽  
Total Dna ◽  
Exonuclease Digestion ◽  
Dwarf Bunt

A linear 7.4-kilobase plasmid designated pTCT was isolated from mycelium of Tilletia controversa Kühn, the causal agent of dwarf bunt of wheat. Linearity was determined by construction of a physical map using eight restriction enzymes and by both 5′ and 3′ exonuclease digestion. pTCT is not homologous to ribosomal genes, nor is it homologous to any sequences in the total DNA of sporidia of Ustilago hordei (isolates 965 and 966) or urediosporelings of Puccinia graminis f.sp. tritici (race C36(48)).

scholarly journals HuluFISH non-denaturing in situ detection of genomic DNA opened by CRISPR-Cas9 Nickase and Exonuclease

2021 ◽  
Author(s):  
Kang Han ◽  
Sheng Liu ◽  
Yongsheng Cheng
Keyword(s):  
In Situ Hybridization ◽  
Genomic Dna ◽  
Genetic Research ◽  
Artificial Chromosome ◽  
Heat Denaturation ◽  
Novel Method ◽  
Exonuclease Digestion ◽  
In Situ Detection ◽  
Novel Design

DNA fluorescence in situ hybridization (FISH) has been widely used in diagnosis and genetic research. Traditional Bacterial artificial chromosome (BAC) or oligonucleotide based probe to detect DNA in situ is only effective when the target is relatively large, usually over 150Kb DNA fragments. And it involves heat denaturation steps to open the DNA for in situ hybridization. The heat process can affect the fine structure of nuclei. Here we reported a novel method based on Cas9 nickase and exonuclease digestion of double strand DNA and permanently mark the DNA in single strand state for FISH. With this novel design, we detected non-repetitive genomic loci as small as 2Kb.

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