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Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.Genome Research Jul 2021Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased...
Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.
PubMed: 34140313
DOI: 10.1101/gr.266551.120 -
Journal of Clinical Microbiology Mar 1990We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the...
We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.
Topics: DNA; DNA, Circular; DNA, Single-Stranded; Electrophoresis, Agar Gel; Eukaryota; Glass; Humans; Microspheres; RNA; RNA, Ribosomal; Silicon Dioxide
PubMed: 1691208
DOI: 10.1128/jcm.28.3.495-503.1990 -
Nature Biotechnology Apr 2024Current N-methyladenosine (mA) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio,...
Current N-methyladenosine (mA) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale mA RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying mA in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark mA mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.
Topics: Animals; Mice; Zebrafish; RNA; RNA, Messenger; Embryonic Stem Cells; Cells, Cultured
PubMed: 37349523
DOI: 10.1038/s41587-023-01831-7 -
Molecules (Basel, Switzerland) Jan 2021Melatonin is a hormone secreted in the pineal gland with several functions, especially regulation of circadian sleep cycle and the biological processes related to it.... (Review)
Review
Melatonin is a hormone secreted in the pineal gland with several functions, especially regulation of circadian sleep cycle and the biological processes related to it. This review evaluates the bioavailability of melatonin and resulting metabolites, the presence of melatonin in wine and beer and factors that influence it, and finally the different benefits related to treatment with melatonin. When administered orally, melatonin is mainly absorbed in the rectum and the ileum; it has a half-life of about 0.45-1 h and is extensively inactivated in the liver by phase 2 enzymes. Melatonin (MEL) concentration varies from picograms to ng/mL in fermented beverages such as wine and beer, depending on the fermentation process. These low quantities, within a dietary intake, are enough to reach significant plasma concentrations of melatonin, and are thus able to exert beneficial effects. Melatonin has demonstrated antioxidant, anticarcinogenic, immunomodulatory and neuroprotective actions. These benefits are related to its free radical scavenging properties as well and the direct interaction with melatonin receptors, which are involved in complex intracellular signaling pathways, including inhibition of angiogenesis and cell proliferation, among others. In the present review, the current evidence on the effects of melatonin on different pathophysiological conditions is also discussed.
Topics: Animals; Anticarcinogenic Agents; Antioxidants; Beer; Circadian Rhythm; Fermentation; Humans; Melatonin; Neuroprotective Agents; Wine
PubMed: 33440795
DOI: 10.3390/molecules26020343 -
Environmental Science & Technology Apr 2010Traditional and new relationships of polychlorinated biphenyl (PCB) distribution among the solid phases, the free aqueous phase, and biolipids are comprehensively... (Review)
Review
Traditional and new relationships of polychlorinated biphenyl (PCB) distribution among the solid phases, the free aqueous phase, and biolipids are comprehensively reviewed using seven well-characterized freshwater and marine sediments polluted with PCBs. The traditional relationship relating free aqueous concentration and biolipid concentration to sediment total organic carbon, compound octanol-water partitioning coefficient, and solid-phase contaminant concentration overestimates measured free aqueous concentrations and biolipid concentrations by mean factors of 8 and 33, respectively. By contrast, relationships based on measured free aqueous phase concentrations or the PCB mass fraction desorbed from sediment provide reasonable predictions of biolipid concentrations. Solid-phase concentration-based predictions perform better when sorption to amorphous organic matter and black carbon (BC) is distinguished. Contrary to previously published relationships, BC sorption appears to be linear for free aqueous PCB-congener concentrations in the picogram to microgram per liter range.
Topics: Geologic Sediments; Polychlorinated Biphenyls; Water Pollutants, Chemical
PubMed: 19961220
DOI: 10.1021/es902325t -
Academic Forensic Pathology Mar 2017Hundreds of synthetic substances have been introduced into the illicit drug market over the last ten years, but none of these drugs has had as poisonous a consequence as... (Review)
Review
Hundreds of synthetic substances have been introduced into the illicit drug market over the last ten years, but none of these drugs has had as poisonous a consequence as the emergence of the synthetic fentanyl analogs. Initially, pharmaceutical grade or illicit fentanyl was mixed with heroin, allegedly to boost the potency of the heroin. Then, the amounts of fentanyl spiked gradually increased until the proportion of fentanyl was greater than the proportion of heroin. Ultimately, many overdose cases began consisting of only fentanyl. The emergence of numerous synthetic fentanyl analogs, including acetylfentanyl, butyrylfentanyl, acrylfentanyl, furanylfentanyl and β-hydroxythiofentanyl, which are manufactured in China, were made available to the illicit drug traffickers over the Internet. In July of 2016, the most potent commercially available opioid, carfentanil, started appearing in illicit drug submissions and medical examiner death investigation cases in Northeast Ohio. Postmortem femoral blood carfentanil concentrations are in the picogram per milliliter (pg/mL) range, which is extremely low, and tests the limits of detection for most analytical forensic toxicology laboratories. The interpretation of these low carfentanil blood concentrations in antemortem and postmortem specimens is made difficult due to the overlap in the concentrations between these specimen types. The presence of these powerful synthetic fentanyl analogs presents a challenge to forensic toxicology laboratories preparing to analyze for these substances.
PubMed: 31239954
DOI: 10.23907/2017.004 -
Astrobiology Dec 2022For exploring nearby stars, let us consider the challenges of a picogram- to nanogram-scale probe to land, replicate, and produce a communications module based on...
For exploring nearby stars, let us consider the challenges of a picogram- to nanogram-scale probe to land, replicate, and produce a communications module based on biominerals at the destination. A billion such probes could be launched for similar cost as a single gram-scale probe. One design is a highly reflective light sail, traveling a long straight line toward the gravitational well of a destination star, and then photo-deflected to the closest nonluminous mass-ideally a planet or moon with exposed liquid water.
PubMed: 36475966
DOI: 10.1089/ast.2022.0008 -
Environmental Chemistry Letters 2016Many analytical techniques have been used to monitor environmental pollutants. But most techniques are not capable to detect pollutants at nanogram levels. Hence, under... (Review)
Review
Many analytical techniques have been used to monitor environmental pollutants. But most techniques are not capable to detect pollutants at nanogram levels. Hence, under such conditions, absence of pollutants is often assumed, whereas pollutants are in fact present at low but undetectable concentrations. Detection at low levels may be done by nano-capillary electrophoresis, also named microchip electrophoresis. Here, we review the analysis of pollutants by nano-capillary electrophoresis. We present instrumentations, applications, optimizations and separation mechanisms. We discuss the analysis of metal ions, pesticides, polycyclic aromatic hydrocarbons, explosives, viruses, bacteria and other contaminants. Detectors include ultraviolet-visible, fluorescent, conductivity, atomic absorption spectroscopy, refractive index, atomic fluorescence spectrometry, atomic emission spectroscopy, inductively coupled plasma, inductively coupled plasma-mass spectrometry, mass spectrometry, time-of-flight mass spectrometry and nuclear magnetic resonance. Detection limits ranged from nanogram to picogram levels.
PubMed: 32214934
DOI: 10.1007/s10311-015-0547-x