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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification.

Ryan R Hansen1, Heather J Avens, Raveesh Shenoy

  • 1Department of Chemical and Biological Engineering, ECCH 111 CB 424, University of Colorado, Boulder, CO 80309, USA.

Analytical and Bioanalytical Chemistry
|July 29, 2008
PubMed
Summary
This summary is machine-generated.

This study presents a novel nonenzymatic assay for quantifying DNA targets on biochips. The method uses polymer growth and fluorescent nanoparticles for sensitive, point-of-care molecular diagnostics.

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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Assay Development

Background:

  • Quantitative analysis of minimal polynucleotide concentrations is crucial for molecular diagnostics.
  • There is a need for high-throughput, nonenzymatic, sensitive, and quantitative assays for point-of-care testing.

Purpose of the Study:

  • To develop a nonenzymatic method for quantifying surface concentrations of labeled DNA targets.
  • To couple regulated polymer growth to biomolecular binding on array-based biochips.
  • To enhance assay compatibility with fluorescence-based detection for point-of-care applications.

Main Methods:

  • A nonenzymatic method was developed using regulated polymer growth on array-based biochips.
  • Polymer film thickness was measured (20-220 nm) and correlated with DNA surface concentrations.
  • Fluorescent nanoparticles were incorporated into polymer films to enhance signal detection.

Main Results:

  • Polymer film thickness showed a logarithmic relationship with labeled DNA surface concentrations over two orders of magnitude.
  • A lower limit of quantitation was achieved at 60 molecules/µm² (approximately 10^6 target molecules).
  • Incorporation of fluorescent nanoparticles significantly increased fluorescent signal for point-of-care detection.

Conclusions:

  • The developed nonenzymatic method enables sensitive and quantitative DNA target detection on biochips.
  • The assay is feasible for point-of-care testing due to its compatibility with fluorescence-based detection.
  • This approach advances molecular diagnostic technology by offering a high-throughput, nonenzymatic quantification method.