Publications by authors named "John S Eid"
A gene-level targeted enrichment method for direct detection of epigenetic modifications is described. The approach is demonstrated on the CGG-repeat region of the FMR1 gene, for which large repeat expansions, hitherto refractory to sequencing, are known to cause fragile X syndrome. In addition to achieving a single-locus enrichment of nearly 700,000-fold, the elimination of all amplification steps removes PCR-induced bias in the repeat count and preserves the native epigenetic modifications of the DNA.
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- Over 40% of male and ~16% of female carriers of the FMR1 premutation allele may develop neurodegenerative disorders, with RNA toxicity from increased mRNA levels being a leading factor in these conditions.
- A long-read sequencing method identified 16 isoforms of FMR1 mRNA, with a significant increase in isoforms Iso10 and Iso10b in premutation carriers.
- These findings point to the potential for relative increases in FMR1 mRNA isoforms contributing to the pathology associated with fragile X-related disorders.
Article Synopsis
- The FMR1 gene contains a repeating CGG sequence, with expansions linked to disorders like fragile X syndrome (over 200 repeats) and fragile X-associated tremor/ataxia syndrome (55-200 repeats).
- Traditional DNA sequencing methods struggle to analyze these expanded repeats, particularly in the full mutation range.
- Single-molecule, real-time (SMRT) sequencing offers a solution by accurately sequencing long, repetitive DNA sequences, revealing unique interactions between CGG repeats and DNA polymerase activity, which could lead to new insights in further research.
A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular.
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- Researchers used advanced microscopy to measure the fluorescent lifetimes of DNA molecules tagged with specific fluorescent dyes.
- They developed a new statistical method to reduce interference from background fluorescence, which typically complicates data collection.
- The study highlights the importance of accounting for background noise to obtain accurate fluorescent lifetime measurements from single molecules.
Article Synopsis
- Individual Cy5 fluorophores attached to DNA were studied using single-molecule spectroscopy to measure their blinking behavior under deoxygenated conditions, revealing spontaneous on/off fluctuations lasting seconds.
- The blinking of Cy5 is influenced by factors such as the donor's proximity, structure, and the presence of dual wavelength excitation light (514 nm and 640 nm).
- By utilizing an alternating laser excitation strategy, researchers can effectively distinguish between the dark states of Cy5 blinking and fluctuations due to FRET, addressing issues with slow blinking that coincide with critical biological time scales.
Single molecule fluorescence resonance energy transfer has been extensively used to measure distance changes and kinetics in various biomolecular systems. However, due to complications involving multiple de-excitation pathways of the dyes, the absolute inter-dye distance information has seldom been recovered. To circumvent this we directly probe the relative variations in the quantum yield of individual fluorophores.
View Article and Find Full Text PDFHeptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy.
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