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Digital Polymerase Chain Reaction Paired with High-Speed Atomic Force Microscopy for Quantitation and Length Analysis
Sean R Koebley1, Andrey Mikheikin1, Kevin Leslie1
1Physics Department, Virginia Commonwealth University, Richmond, Virginia 23284, United States.
ACS Nano
|November 10, 2020
Summary
This study introduces a novel digital PCR and high-speed AFM method to accurately measure DNA length variations. This technique aids in diagnosing diseases like acute myeloid leukemia by precisely quantifying genetic polymorphisms.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- DNA length polymorphisms are crucial biomarkers in various diseases.
- Accurate quantification of these polymorphisms, especially in mixed populations, presents significant challenges due to their repetitive nature.
Purpose of the Study:
- To develop a simple, rapid, and flexible method for quantifying DNA length and proportion.
- To address the limitations in measuring DNA length polymorphisms in complex genetic backgrounds.
Main Methods:
- Combined digital PCR (dPCR) with high-speed atomic force microscopy (HSAFM).
- Direct imaging and sizing of individual DNA amplicons from dPCR partitions.
- Focused on internal tandem duplications (ITDs) in the FLT3 gene associated with acute myeloid leukemia.
Main Results:
- The dPCR-HSAFM method accurately determined variant length and allele frequency for FLT3-ITDs.
- Successfully quantified variants down to 5% in mixed samples.
- Analyzed over 1.5 million amplicons from cell lines and clinical samples.
Conclusions:
- dPCR-HSAFM offers high-throughput, single-molecule resolution for DNA polymorphism analysis.
- Represents a significant advancement in HSAFM applications.
- Provides a powerful new tool for diagnosing genetic length polymorphisms.
