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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.
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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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Facile SNP detection using bifunctional, cross-linking oligonucleotide probes.

Xiaohua Peng¹, Marc M Greenberg

¹Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.

Nucleic Acids Research

|February 19, 2008

Summary

This study introduces a new method for detecting specific DNA sequences with high accuracy. The technique uses oligonucleotide probes and a cross-linking reaction for sensitive, single-nucleotide-level DNA detection without PCR.

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Area of Science:

Molecular Biology
Biochemistry
Analytical Chemistry

Background:

Accurate detection of specific DNA sequences is crucial for various biological and diagnostic applications.
Existing methods may lack sensitivity or single-nucleotide discrimination capabilities.
Developing novel assays for oligonucleotide detection is an ongoing area of research.

Purpose of the Study:

To develop a facile and sensitive method for detecting specific oligonucleotide sequences.
To achieve single nucleotide discrimination in DNA sequence detection.
To establish a robust assay for probing target sequences in plasmid DNA.

Main Methods:

Utilized bifunctional oligonucleotide probes with internal modified pyrimidines and 5'-biotin.
Employed a hybridization strategy combined with an oxidative cross-linking reaction.
Detected target sequences using fluorescence spectroscopy with avidin and horseradish peroxidase conjugates in a microtiter plate assay.

Main Results:

Achieved sensitive detection of target DNA sequences with as little as 250 fmol without using PCR.
Demonstrated single nucleotide discrimination with a ratio approaching 200:1.
Successfully discriminated between 16-nt long sites in plasmid DNA differing by a single nucleotide.

Conclusions:

The developed method offers a sensitive and specific approach for oligonucleotide detection.
The assay provides high single nucleotide discrimination, valuable for genetic analysis.
This technique has the potential for probing any target sequence containing 2'-deoxyadenosine.