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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
To overcome issues surrounding background noise, together with the amounts of sequencing data and starting material required for ChIP-sequencing, two new methods have been developed. The first is Cleavage Under Targets and Release Under Nuclease (or CUT&RUN) which was first established in 2017 as a more efficient and cost-effective approach for analysing molecule binding sites (31). This approach uses a similar antibody immunoprecipitation method as suggested previously, but then exposes the DNA to an enzyme that can recognise the antibody and cut the DNA around it. The resultant DNA fragments are then purified and can be used for making DNA sequencing libraries through standardised protocols. Importantly, the enzyme cutting process occurs in a highly specific and effective manner and without the need for prior fixation of the tissue or cell sample, which collectively, greatly reduces both background noise and the potential discovery of false positives (32). Indeed, it is suggested that reliable data can be obtained from as little as 100–1,000 cells (31).
The Journey through the Gene: a Focus on Plant Anti-pathogenic Agents Mining in the Omics Era
Published in Mahendra Rai, Chistiane M. Feitosa, Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
José Ribamar Costa Ferreira-Neto, Éderson Akio Kido, Flávia Figueira Aburjaile, Manassés Daniel da Silva, Marislane Carvalho Paz de Souza, Ana Maria Benko-Iseppon
One of the most critical features in molecular genetics is the ability of DNA sequencing. At the beginning of the DNA sequencing era, the applied methodologies involved high cost/million bases and a long time/run necessary to obtain good quality sequence data. These factors became almost impossible to study large genomes. With the emergence of Second-Generation Sequencing (SGS; previously known as Next Generation Sequencing or NGS) technologies, this scenario started to be modified.
Prenatal Diagnosis and Screening for Aneuploidy
Published in Vincenzo Berghella, Obstetric Evidence Based Guidelines, 2022
Sarah Harris, Angie Jelin, Neeta Vora
Although promising, this new technology has many challenges that are still being uncovered, including long turnaround time for results, incomplete data on prenatal phenotypes and sequence variants, ethical issues related to incidental findings, the large amount of data and need for reanalysis, and a high cost associated with extensive DNA sequencing [61].
When Less is More: Lessons for Expanded Carrier Screening from Newborn Sequencing Research
Published in The American Journal of Bioethics, 2023
Such a vision has been articulated multiple times by Francis Collins, the former director of both the National Institutes of Health and the Human Genome Project, including in a 2014 op-ed in the Wall Street Journal. “Over the course of the next few decades” Collins writes, “the availability of cheap, efficient DNA sequencing technology will lead to a medical landscape in which each baby’s genome is sequenced, and that information is used to shape a lifetime of personalized strategies for disease prevention, detection and treatment.” Collins doesn’t specify here whether this universal sequencing should happen via newborn screening programs, which in the US are effectively mandatory because infants’ blood spots are collected at the hospital without parental permission (in some countries, parents are asked for their consent), or as a part of routine pediatric care (a context where parental informed consent or refusal should be possible).
Endophthalmitis: Microbiology and Organism Identification Using Current and Emerging Techniques
Published in Ocular Immunology and Inflammation, 2023
Christine L. Tan, Harsha Sheorey, Penelope J. Allen, Rosie C. H. Dawkins
On the other hand, a 16S rRNA gene (for bacteria) and 18S rRNA gene (for fungi and parasites) based PCR assay with nucleic acid sequencing is useful for an unknown organism. 16S/18S rRNA gene contains conserved regions useful for the design of broad-range PCR primers that can amplify various fragments of the 16S/18S rRNA gene from organisms that may be present in the specimen. This is a multistep process that includes extracting the DNA, sequencing it, and then running the sequence/signature in a known database to get a match with a known organism. Each step requires different expertise and a lab will have to put a specific team together. Typically, the last step is outsourced to an external provider with computer skills to analyze the data. So again, it defeats the purpose of “rapid” diagnosis by molecular means. This assay is available only in a handful of reference laboratories and is very expensive at this stage. The best use of this assay is when we see the organism (in Gram’s stain or histology) but do not isolate it, e.g. after antibiotics are given or very occasionally organisms that are difficult/slow to grow or non-cultivable. A 16 s or 18 s rRNA PCR cannot differentiate contamination from true infection and being very sensitive, it picks up contaminants (that can be at any level from collection and handling in the laboratory to processing). Commercial “all in one” assays are being developed and may be available in the near future.
STOX1 promotor region -922 T > C polymorphism is associated with Early-Onset preeclampsia
Published in Journal of Obstetrics and Gynaecology, 2022
Seyda Akin, Ergun Pinarbasi, Aslihan Esra Bildirici, Nilgun Cekin
DNA sequencing was conducted by Sanger sequencing method. A 3–5-μL aliquot of PCR product (collected directly or from re-amplification of excised SSCA bands) was used in a standard protocol for fluorescently labelled dideoxy-nucleotides (BigDye, Applied Biosystems, Life Technologies), with injection into a capillary electrophoresis instrument (ABI 3500, Life Technologies) for separation and detection. The sequences obtained were compared with the reference sequence NC 000017 (www.ncbi.nlm.nih.gov), and deviations were recorded as mutations or polymorphisms. Chromas Lite 2.6.6 software was used to display the results and to scan the changes in the array. Also, DNA sequencing was employed using the next generation sequencing system Illumina technology. IGV 2.6.3 (Archived) program was utilised to display the results and to observe the changes in the array.