scholarly journals Selective chemical autoligation on a double-stranded DNA template

1994 ◽  
Vol 22 (23) ◽  
pp. 5076-5078 ◽  
Author(s):  
Mathias K. Herrlein ◽  
Robert L. Letsinger
Keyword(s):  
Double Stranded Dna ◽  
Selective Chemical ◽  
Dna Template

scholarly journals Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

2020 ◽  
Author(s):  
Taylor L. Mighell ◽  
Andrew Nishida ◽  
Brendan L. O’Connell ◽  
Caitlin V. Miller ◽  
Sally Grindstaff ◽  
...  
Keyword(s):  
Low Cost ◽  
Targeted Sequencing ◽  
High Input ◽  
Selective Enrichment ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Fold Enrichment ◽  
Gc Bias ◽  
Dna Template

AbstractTargeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive GC sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogramed guide RNAs to direct CRISPR-Cas12a cleavage of double stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7,176 guides targeting 3.5 megabases of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 megabases. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for SNVs, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double stranded DNA template or genome.

scholarly journals Functional Conservation and Specialization among Eukaryotic Anti-Silencing Function 1 Histone Chaperones

2005 ◽  
Vol 4 (9) ◽  
pp. 1583-1590 ◽  
Author(s):  
Beth A. Tamburini ◽  
Joshua J. Carson ◽  
Melissa W. Adkins ◽  
Jessica K. Tyler
Keyword(s):  
Growth Defect ◽  
Histone Chaperones ◽  
Checkpoint Protein ◽  
Functional Conservation ◽  
Dna Damaging Agents ◽  
Double Stranded Dna ◽  
Dna Template ◽  
Nuclear Processes ◽  
Insight Into

ABSTRACT Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with Drosophila Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of Saccharomyces cerevisiae lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the PHO5 gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1's role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.

scholarly journals Properties of mitochondrial DNA polymerase in mitochondrial DNA synthesis in yeast.

1995 ◽  
Vol 42 (3) ◽  
pp. 317-324 ◽  
Author(s):  
T K Biswas ◽  
P Sengupta ◽  
R Green ◽  
P Hakim ◽  
B Biswas ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mt Dna ◽  
Double Stranded Dna ◽  
Dna Template ◽  
Mitochondrial Dna Polymerase

Mitochondrial DNA polymerase from Saccharomyces cerevisiae, purified 3500 fold, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three polypeptides. The major 150 kDa polypeptide was probably the catalytic subunit of the mitochondrial (mt) DNA polymerase and the other two polypeptides could be either proteolytic cleavage products of the polymerase, other subunits of the enzyme or protein contaminants. The mtDNA polymerase preferred an A+T-rich DNA template and did not require any RNA primer for DNA synthesis, at least under in vitro reaction conditions. It showed higher processivity on a double-stranded linear DNA template than on a single-stranded circular DNA template, and was capable of synthesizing at least about 1200 nucleotide primer-extended products without any major pause on a double-stranded DNA template.

Quaternary Polymorphism in Helicases and the DnaB.DnaC Complex

2000 ◽  
Vol 6 (S2) ◽  
pp. 272-273
Author(s):  
L.E. Donate ◽  
M. Bárcena ◽  
O. Llorca ◽  
N. Dixon ◽  
J.M. Carazo
Keyword(s):  
Molecular Weight ◽  
Chromosomal Replication ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Partner Protein ◽  
Total Molecular Weight ◽  
3D Volume ◽  
Dna Template ◽  
Hydrolysis Of

Strand separation in double stranded DNA is achieved in vivo by a class of enzymes called helicases in a process fuelled by hydrolysis of nucleoside triphosphates. DnaB is the major replicative helicase in E.coli. For chromosomal replication to initiate, DnaB needs to interact with a partner protein, namely DnaC, which after properly loading DnaB onto the DNA template at the origin of replication is subsequently released from the complex. DnaB turns to be functionally active as a helicase only after DnaC has been released from the complex. The native DnaB is a homohexamer of molecular weight 318 kD. In the presence of ATP and Mg2+, the hexameric DnaB has been shown to form a complex with six molecules of DnaC (total molecular weight of the complex: 480 kD).The reconstructed 3D volume of the DnaB hexamer obtained from frozen-hydrated specimens showed the DnaB oligomer as a particle possessing three-fold rather than six-fold symmetry, despite DnaB being made up by six identical subunits.

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