A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum

Urdea, Mickey S.; Running, Joyce A.; Horn, Thomas; Clyne, Jennifer; Ku, Lailing; Warner, Brian D.

doi:10.1016/0378-1119(87)90189-2

Cited by 82 publications

(49 citation statements)

References 22 publications

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“…The limit of detection is approximately 10 4 insulin mRNA molecules. The optimal limit of detection for the bDNA assay is approximately 10 3 molecules (14), but the insulin mRNA assay is limited by a suboptimal number of capture and label probes because of the small size of the mouse insulin II mRNA.…”

Section: Resultsmentioning

confidence: 99%

Regulation of insulin preRNA splicing by glucose

Wang

Shen

Najafi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Glucose tightly regulates the synthesis and secretion of insulin by ␤ cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single ␤ cell (␤TC3 cells or mouse islets), whereas 1 million non-insulinproducing ␣ cells (␣TC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from ␤ cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up Ϸ2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription.Insulin, the key signal for energy storage, is synthesized and secreted by the ␤ cells of the pancreatic islets of Langerhans in mammals. Insulin synthesis and secretion is tightly regulated by glucose, which serves as a signal of the energy state of the organism. Synthesis of mature insulin is a multistep process and glucose regulates synthesis at several of these steps, including transcription (1), mRNA stabilization (2), translation (3, 4), and processing proinsulin to insulin (5).The level of insulin mRNA available in the cytoplasm for translation to preproinsulin depends on the relative rates of insulin mRNA production and degradation. Glucose regulates both insulin gene transcription and insulin mRNA degradation (1, 2). Transcription rate alone, however, does not determine the production rate of cytoplasmic insulin mRNA. Prior to export from the nucleus, preRNAs are spliced and a 5Ј m 7 GpppN cap and 3Ј adenosines are added. These processes are not independent: splicing accelerates polyadenylylation, and both are important for nuclear export (6-8).Insulin preRNA in most species contains two introns. The sequences of the introns show little evidence of evolutionary conservation, but the positions of the introns are highly conserved (9, 10). The length of intron 2 varies widely among species, but intron 1 is generally small (118 ...

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Section: Resultsmentioning

confidence: 99%

Regulation of insulin preRNA splicing by glucose

Wang

Shen

Najafi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…The problems associated with PCR are not an issue for signal amplification methods that work by producing reporter molecules in response to the presence of a target, rather than by amplifying the target (1,(11)(12)(13)(14). The amounts of reporter molecules generated by signal amplification methods are proportional to the amounts of target, leading to simple quantitative analysis.…”

Section: Discussionmentioning

confidence: 99%

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Hall

Eis

Law

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

231

151

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The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10 7 reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

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“…Different types of polymeric matrices comprised of DNA itself have been described (20,27,28). These polymeric or dendritic DNA structures have been used to amplify radioactive or fluorescent signals by direct incorporation of label in hybridization-based detection (28)(29)(30). In the bDNA approach, DNA-based polymeric matrices have been used as a platform to attach a significant number of alkaline phosphatase molecules that provided amplification of the chemiluminescent signal for detecting very low viral copy numbers (9).…”

Section: Resultsmentioning

confidence: 99%

Signal amplification through nucleotide extension and excision on a dendritic DNA platform

Capaldi

Getts²,

Jayasena³

2000

Nucleic Acids Research

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Techniques that provide strong signal amplification are useful in diagnostic applications, especially in detecting low concentrations of non-amplifiable target molecules. A versatile and strong signal amplification method based on activities of a DNA polymerase to generate high concentrations of pyrophosphate (PPi) is described. The generation of PPi is catalyzed by nucleotide extension and excision activities of a DNA polymerase on an oligonucleotide cassette. The signal is generated upon enzymatic conversion of PPi to ATP and ATP levels subsequently detected with firefly luciferase. Bioluminesence produced by an oligonucleotide cassette consisting of just two polymerase reaction sites is sufficient to detect them at low attomole levels. The attachment of a large number of these oligonucleotide cassettes to DNA dendrimers enabled the detection of such polyvalent substrate molecules at low zeptomole (10 -21 mol) concentrations. The extent of signal amplification obtained with dendrimer substrates is comparable to exponential target amplifications provided by nucleic acid amplification methods. The attachment of such PPi-generating dendritic DNA platforms to ligands that mediate target recognition would potentially permit detection of extremely low concentrations of analytes in diagnostic assays.

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A novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum

Cited by 82 publications

References 22 publications

Regulation of insulin preRNA splicing by glucose

Regulation of insulin preRNA splicing by glucose

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Signal amplification through nucleotide extension and excision on a dendritic DNA platform

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