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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
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Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...
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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis
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Continuously tunable nucleic acid hybridization probes.

Lucia R Wu1, Juexiao Sherry Wang1,2, John Z Fang1

  • 1Department of Bioengineering, Rice University, Houston, Texas, USA.

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|October 20, 2015
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Summary
This summary is machine-generated.

This study introduces a novel method to fine-tune nucleic acid probe performance on-the-fly. This technique optimizes probe binding and signal in complex biological samples, improving assay accuracy.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • * Designing nucleic acid probes for assays often requires iterative optimization for specificity and sensitivity.
  • * Multiplexed assays present challenges in balancing probe performance simultaneously.

Purpose of the Study:

  • * To present a novel, on-the-fly method for tuning probe affinity and selectivity.
  • * To enable independent adjustment of hybridization yield for multiple probes in multiplexed assays.

Main Methods:

  • * Adjusting the stoichiometry of auxiliary species to tune probe effective free energy.
  • * Applying stoichiometric tuning to Nanostring nCounter platform for multiplexed assays.
  • * Demonstrating tunable hybrid capture of mRNA and quantitation of RNA variants.

Main Results:

  • * Achieved near-continuous tuning of probe effective free energy.
  • * Enforced uniform capture efficiency for diverse DNA molecules (0-100% GC content).
  • * Maximized signal difference for single-nucleotide variants and performed multiplexed RNA quantitation.

Conclusions:

  • * The presented method allows for decoupled adjustment of probe hybridization yields.
  • * Stoichiometric tuning is effective for optimizing multiplexed nucleic acid assays, including for FFPE samples.