Label-Free and Homogeneous Sensing Platform with Template-Independent Cascade Amplification Strategy for Genome-Wide...

Area of Science:

• Epigenetics
• Molecular Biology
• Biochemistry

Background:

• 5-Hydroxymethylcytosine (5hmC) is a crucial epigenetic DNA modification implicated in cancer and neurodegenerative diseases.
• Distinguishing 5hmC from 5-methylcytosine (5mC) is challenging due to their structural similarity, often requiring complex or damaging methods.

Purpose of the Study:

• To develop a sensitive, homogeneous, label-free, and genome-wide detection platform for 5hmC.
• To overcome limitations of existing methods, enabling efficient and accurate 5hmC quantification.

Main Methods:

• A template-independent cascade amplification strategy was employed, integrating terminal transferase (TdT)-mediated amplification with APE1-driven cyclic cleavage.
• The assay utilizes a signal probe with an apurinic/apyrimidinic (AP) site, eliminating the need for DNA separation steps.

Main Results:

• The platform achieved a low detection limit (6.34 × 10^-17 M) and could detect as little as 0.099% 5hmC in the presence of 5mC and cytosine.
• High sensitivity was demonstrated at the cellular level (2.33 × 10^-5 ng/μL), distinguishing various tumor cell lines and tissues.
• The method showed high accuracy in differentiating cancer patients from healthy individuals (AUC=0.993) with a 3.6-fold cost reduction compared to ELISA kits.

Conclusions:

• The developed sensing platform provides a robust and efficient tool for genome-wide 5hmC detection.
• This method holds significant potential for applications in tumor liquid biopsy and epigenetic marker screening.
• The platform offers a cost-effective and sensitive alternative for epigenetic analysis in disease diagnostics.