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Related Concept Videos

FISH - Fluorescent In-situ Hybridization02:07

FISH - Fluorescent In-situ Hybridization

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,...
Labeling DNA Probes03:31

Labeling DNA Probes

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...
Southern Blot02:57

Southern Blot

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.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
In-situ Hybridization02:31

In-situ Hybridization

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.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...

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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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Hybridization-sensitive fluorescent DNA probe with self-avoidance ability.

Shuji Ikeda1, Takeshi Kubota, Mizue Yuki

  • 1RIKEN Advanced Science Institute, Wako, Saitama, 351-0198, Japan.

Organic & Biomolecular Chemistry
|January 22, 2010
PubMed
Summary
This summary is machine-generated.

New fluorescent probes using unnatural bases 2'-deoxyinosine (I) and N(4)-ethyl-2'-deoxycytidine (E) reduce self-dimerization and background noise. This improves signal-to-noise ratios for sensitive target detection in molecular imaging.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Nucleic Acid Chemistry

Background:

  • Hybridization-sensitive fluorescent probes often suffer from high background signals due to probe self-dimerization.
  • This self-dimerization interferes with effective fluorescence quenching before target hybridization.

Purpose of the Study:

  • To develop novel hybridization-sensitive fluorescent probes that minimize self-dimerization.
  • To enhance the signal-to-noise ratio for improved detection of target nucleic acid sequences.

Main Methods:

  • Synthesized new fluorescent probes (IE probes) incorporating unnatural nucleotides: 2 -deoxyinosine (I), N(4)-ethyl-2 -deoxycytidine (E), and a doubly thiazole orange-labeled nucleotide (D(514)).
  • Investigated probe thermostability and absorption spectra to assess hybridization-dependent changes.
  • Evaluated the ratio of fluorescence intensities (I(hybrid)/I(nonhybrid)) before and after hybridization.

Main Results:

  • IE probes exhibited low thermostability, preventing self-dimerization and reducing background fluorescence.
  • Hybridization induced a shift in absorption maxima, indicating effective dye-dye excitonic interaction.
  • Replacing guanine and cytosine with I and E significantly improved the I(hybrid)/I(nonhybrid) ratio.
  • Replacing adjacent E bases near D(514) with cytosine restored fluorescence intensity in hybridized probes.

Conclusions:

  • The incorporation of I and E bases effectively suppresses probe self-dimerization, leading to lower background signals.
  • Enhanced dye-dye excitonic interaction in IE probes improves fluorescence signal upon hybridization.
  • These optimized probes facilitate the design of longer sequences for sensitive mRNA imaging applications.