Amplification-Free Quantification of Endogenous Mitochondrial DNA Copy Number Using Solid-State Nanopores.

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• Mitochondrial DNA (mtDNA) quantification is vital for understanding diseases like cancer and neurodegenerative disorders.
• Conventional methods often use amplification, which can introduce bias and lack diagnostic precision.
• Solid-state nanopores offer label-free, single-molecule detection without amplification.

Purpose of the Study:

• To develop an amplification-free method for mtDNA quantification using solid-state nanopores and machine learning.
• To specifically detect and quantify mtDNA from genomic DNA background.
• To enable accurate mtDNA quantification for clinical diagnostics and point-of-care applications.

Main Methods:

• Utilized solid-state nanopores for label-free, single-molecule detection of DNA.
• Employed selective degradation of genomic DNA (gDNA) using exonuclease V.
• Applied a support vector machine (SVM) model for mtDNA classification and quantification.
• Performed synchronous electro-optical sensing to investigate mtDNA-protein complexes.

Main Results:

• Successfully distinguished and quantified native mtDNA from gDNA fragments.
• Achieved high classification accuracy in identifying mtDNA translocations.
• Demonstrated quantification of endogenous mtDNA in cancer cell lines and blood cells.
• Detected mtDNA at picomolar levels, suitable for low-abundance samples.
• Identified mtDNA as complexed with packaging proteins via electro-optical sensing.

Conclusions:

• The developed method provides robust and accurate amplification-free mtDNA quantification.
• Solid-state nanopores combined with machine learning offer a sensitive and specific approach.
• This technique minimizes sample preparation and has potential for point-of-care diagnostics.
• The findings highlight the structural distinctiveness of native mtDNA for detection.