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Related Concept Videos

Labeling DNA Probes03:31

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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.
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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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Strand-invading linear probe combined with unmodified PNA.

Hiroyuki Asanuma1, Rie Niwa1, Mariko Akahane1

  • 1Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan.

Bioorganic & Medicinal Chemistry
|July 11, 2016
PubMed
Summary

Researchers developed a fluorescent DNA probe for labeling double-stranded DNA. This probe, combined with peptide nucleic acid (PNA), efficiently invades DNA and allows for sensitive detection, even at DNA termini.

Keywords:
DNALinear probePNAStrand invader

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Biotechnology

Background:

  • Fluorescent labeling of double-stranded DNA (dsDNA) is crucial for molecular biology applications.
  • Developing probes that can efficiently and specifically target dsDNA remains a challenge.
  • Existing methods often require DNA denaturation or exhibit high background fluorescence.

Purpose of the Study:

  • To develop a novel linear probe for efficient and specific fluorescent labeling of dsDNA.
  • To investigate the mechanism of strand invasion by the probe and peptide nucleic acid (PNA) complex.
  • To enhance probe stability and reduce background emission for sensitive dsDNA detection.

Main Methods:

  • Synthesis of a linear DNA probe incorporating ethynylperylene fluorophores via a d-threoninol scaffold.
  • Utilizing an unmodified peptide nucleic acid (PNA) as an invader.
  • Employing heat-shock treatment to facilitate strand invasion and double duplex formation.
  • Gel-shift assays to analyze probe-DNA-PNA interactions.
  • Synthesis of a modified probe with anthraquinones and ethynylperylene for improved performance.

Main Results:

  • The ethynylperylene-modified DNA probe showed minimal disruption of DNA hybridization.
  • The linear probe exhibited self-quenching in the absence of target DNA.
  • A combination of the linear probe and PNA successfully invaded dsDNA upon heat shock, forming a double duplex.
  • The enhanced probe with anthraquinones and ethynylperylene enabled highly sensitive detection of internal dsDNA sequences.
  • The probe-PNA pair demonstrated invasion at the terminus of long dsDNA at 40°C and 100mM NaCl.

Conclusions:

  • Efficient fluorescent labeling of dsDNA is achievable using a linear probe and PNA invader.
  • Heat-shock treatment is effective in promoting strand invasion for dsDNA detection.
  • The developed probe system offers high sensitivity and stability for dsDNA sequence detection.