PS-Modified DNA Barcode PCR for Sensitive Disease Readout
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Solution Overview
Problem
Existing methods for detecting DNA barcodes, particularly those with phosphorothioate modifications, face challenges in amplification due to weak polymerase association, leading to difficulties in detecting diseases like cancer with high sensitivity and stability.
Innovation Solution
A method involving the use of phosphorothioated DNA barcodes of specific lengths (36 to 72 nucleotides) with priming sequences and optional unique molecular identifiers, amplified via PCR, to enhance detection in biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If phosphorothioate modifications are added to DNA barcodes to increase half-life, then stability is improved, but PCR amplification efficiency deteriorates
Solution Approach 1:
The patent applies local quality by modifying only specific positions in the DNA barcode sequence with phosphorothioate bonds rather than uniformly modifying the entire sequence. This selective modification at critical positions maintains stability while preserving PCR amplification capability at unmodified positions.
Solution Approach 2:
The patent changes the chemical parameter of the DNA backbone by introducing phosphorothioate modifications at specific positions. This parameter change increases nuclease resistance and half-life while the patent optimizes the number and position of modifications to maintain amplification efficiency.
2Measurement precision
If DNA barcode length is increased to improve detection sensitivity, then measurement precision is improved, but amplification efficiency deteriorates
Solution Approach 1:
The patent optimizes the length parameter of the DNA barcode to a specific range (36-72 nucleotides) that balances detection sensitivity with amplification efficiency. This parameter optimization ensures the barcode is long enough for specific detection but not so long that amplification becomes inefficient.
3Reliability
If phosphorothioate modifications are increased to enhance stability, then reliability is improved, but polymerase association deteriorates
Solution Approach 1:
The patent applies local quality by strategically placing phosphorothioate modifications at specific positions within the DNA barcode sequence rather than uniform modification. This localized approach protects critical regions from degradation while leaving other regions accessible to polymerase for efficient amplification.
Solution Approach 2:
The patent applies partial action by using a limited number of phosphorothioate modifications (e.g., 2-5 positions) rather than complete modification of the sequence. This partial modification provides sufficient stability enhancement while maintaining adequate polymerase association for amplification.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method improves the amplification efficiency and detection sensitivity of phosphorothioated DNA barcodes, enabling accurate disease diagnosis and treatment monitoring through quantification of DNA barcode levels.
Implementation Method 1
the DNA barcode sequence in the biological sample is amplified and detected
Data Source
AI summary
Provided herein are methods for detecting the presence or progression of a disease in a subject as well as a method for determining the efficacy of a therapy for the disease by administering to the subject a probe comprising a phosphorothioated DNA barcode sequence, wherein the DNA barcode sequence comprises about 36 nucleotides to about 72 nucleotides, and wherein the DNA barcode is released when the disease is present in the subject; obtaining a biological sample containing the released DNA barcode from the subject; and amplifying and detecting the DNA barcode in the biological sample.