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Color-coded molecular beacons for multiplex PCR screening assays

review of the manuscript. Author Contributions Conceptualization: Salvatore A. E. Marras, Sanjay Tyagi, Dan-Oscar Antson, Fred Russell Kramer. Formal analysis: Salvatore A. E. Marras, Sanjay Tyagi, Dan ... -Oscar Antson, Fred Russell Kramer. Funding acquisition: Sanjay Tyagi, Fred Russell Kramer. Investigation: Salvatore A. E. Marras, Dan-Oscar Antson. Methodology: Salvatore A. E. Marras, Sanjay Tyagi

Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5...

Spermatid Cyst Polarization in Drosophila Depends upon apkc and the CPEB Family Translational Regulator orb2

Mature Drosophila sperm are highly polarized cells—on one side is a nearly 2 mm long flagellar tail that comprises most of the cell, while on the other is the sperm head, which carries the gamete's genetic information. The polarization of the sperm cells commences after meiosis is complete and the 64-cell spermatid cyst begins the process of differentiation. The spermatid nuclei...

Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status

RNA flow cytometry (FISH-Flow) achieves high-throughput measurement of single-cell gene expression by combining in-situ nucleic acid hybridization with flow cytometry. We tested whether antigen-specific T-cell responses detected by FISH-Flow correlated with latent tuberculosis infection (LTBI), a condition affecting one-third of the world population. Peripheral-blood mononuclear...

Fusion FISH Imaging: Single-Molecule Detection of Gene Fusion Transcripts In Situ

Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional...

Interactions between the FTO and GNB3 Genes Contribute to Varied Clinical Phenotypes in Hypertension

Background The genes FTO and GNB3 are implicated in essential hypertension but their interaction remains to be explored. This study investigates the role of interaction between the two genes in the pathophysiology of essential hypertension. Methods/Principal Findings In a case-control study comprising 750 controls and 550 patients, interaction between the polymorphisms of FTO and...

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNAPhe form functionally active ternary complex that has properties...

Stochastic mRNA Synthesis in Mammalian Cells

Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number...

Efficiencies of fluorescence resonance energy transfer and contact‐mediated quenching in oligonucleotide probes

Salvatore A. E. Marras 0 Fred Russell Kramer 0 Sanjay Tyagi 0 0 Public Health Research Institute , 225 Warren Street, Newark, NJ 07103, USA An important consideration in the design of

tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells

When oligonucleotide probes are microinjected into cells to image the distribution of RNAs, they are rapidly sequestered into the nucleus. As a result, it is difficult to detect mRNAs in the cytoplasm of living cells. We were able to overcome this process by attaching tRNA transcripts to the probes. We show that when fluorescently labeled tRNAs, tRNAs with extensions at their 5...

Real‐time measurement of in vitro transcription

We have developed a simple method to measure RNA synthesis in real time. In this technique, transcription reactions are performed in the presence of molecular beacons that possess a 2′‐O‐methylribonucleotide backbone. These probes become fluorescent as they hybridize to nascent RNA during the course of synthesis. We found that molecular beacons synthesized from natural...