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Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.
This article was published in the following journal.
Name: Analytical biochemistry
RNA amplification has extensive applications on biochemistry and its related fields. Here, we present an isothermal strategy named rolling circle reverse transcription-mediated RNA amplification (RCRT...
Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was estab...
A new, facile, low-cost, and highly sensitive method for detection of Listeria monocytogenes involving a combination of asymmetric polymerase chain reaction (aPCR) and rolling circle amplification (RC...
For early detection and diagnosis of gastric cancer, a novel, highly sensitive detection of microRNA-378 was developed through rolling circle amplification and DNA-templated silver nanoclusters as a l...
This work reports on a method for determination of lead(II) ion in environmental water samples. A Pb-specific DNAzyme immobilized on magnetic beads was coupled to rolling circle amplification (RCA) an...
The purpose of this study is to investigate the acute effects of foam rolling on elbow joint sensorimotor function with healthy participants . Study group will receive foam rolling exercis...
The purpose of this study is to investigate the long-term effects of foam rolling on elbow proprioception, strength and functional motor performance with healthy participants . Study group...
Older people residing in nursing homes (NH) are frequently polymedicated and often prescribed potentially inappropriate medications. Deprescribing has been proposed as a way to reduce the ...
The task of caring for an individual with Alzheimer's disease is often carried out by elderly caregivers who, due to their motor and psychosocial alterations due to the aging process, may ...
Delayed-onset muscle soreness (DOMS) is an acute micro-trauma or inflammatory response experienced in the most runners; and, it concurrently impairs athletic performance and may increase r...
A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes. It occurs naturally via the excision of a copy of the repeating sequence from the chromosome and its extrachromosomal replication in a plasmid, or via the production of an RNA transcript of the entire repeating sequence of ribosomal RNA followed by the reverse transcription of the molecule to produce an additional copy of the original DNA sequence. Laboratory techniques have been introduced for inducing disproportional replication by unequal crossing over, uptake of DNA from lysed cells, or generation of extrachromosomal sequences from rolling circle replication.
Movement of tethered, spherical LEUKOCYTES along the endothelial surface of the microvasculature. The tethering and rolling involves interaction with SELECTINS and other adhesion molecules in both the ENDOTHELIUM and leukocyte. The rolling leukocyte then becomes activated by CHEMOKINES, flattens out, and firmly adheres to the endothelial surface in preparation for transmigration through the interendothelial cell junction. (From Abbas, Cellular and Molecular Immunology, 3rd ed)
Laboratory techniques that involve the in-vitro synthesis of many copies of DNA or RNA from one original template.
A technique for identifying specific DNA sequences that are bound, in vivo, to proteins of interest. It involves formaldehyde fixation of CHROMATIN to crosslink the DNA-BINDING PROTEINS to the DNA. After shearing the DNA into small fragments, specific DNA-protein complexes are isolated by immunoprecipitation with protein-specific ANTIBODIES. Then, the DNA isolated from the complex can be identified by PCR amplification and sequencing.
The arrangement of two or more amino acid or base sequences from an organism or organisms in such a way as to align areas of the sequences sharing common properties. The degree of relatedness or homology between the sequences is predicted computationally or statistically based on weights assigned to the elements aligned between the sequences. This in turn can serve as a potential indicator of the genetic relatedness between the organisms.
Bioinformatics is the application of computer software and hardware to the management of biological data to create useful information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied...
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Recombinant DNA is the formation of a novel DNA sequence by the formation of two DNA strands. These are taken from two different organisms. These recombinant DNA molecules can be made with recombinant DNA technology. The procedure is to cut the DNA of ...