Circular DNA Template Construction for Bias-Free Sequencing
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Solution Overview
Problem
Current gene sequencing methods are labor-intensive, costly, and prone to bias, making them inefficient for high accuracy and rapid detection of genetic mutations.
Innovation Solution
A method is developed to construct a circular template by preparing partially double-stranded linear DNA molecules with single-stranded protruding portions, which are then ligated to form partially double-stranded circular DNA molecules, enabling circular amplification and sequencing with reduced bias and cost.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If current library construction and sequencing methods are used, then DNA sequencing can be performed, but the process is labor intensive and high cost
Solution Approach 1:
The patent converts linear DNA molecules into circular templates through ligation of complementary overhangs. This circular structure enables continuous sequencing reactions without the need for linear fragment assembly, thereby reducing labor intensity and time consumption while maintaining sequencing accuracy.
Solution Approach 2:
The circular template structure allows the DNA molecule to serve itself as a continuous sequencing template. The complementary overhangs self-ligate to form circles that can be directly used for sequencing, eliminating the need for complex library construction steps and reducing overall process complexity.
2Measurement precision
If current library construction and sequencing methods are used, then DNA sequencing can be performed, but amplification bias is produced
Solution Approach 1:
The patent extracts and utilizes only the essential features of DNA sequencing (the template and primer binding sites) while eliminating the problematic amplification step entirely. By using circular templates with complementary overhangs that self-ligate, the method achieves sequencing without introducing amplification bias, thereby improving measurement precision.
3Measurement precision
If current library construction and sequencing methods are used, then DNA sequencing can be performed, but the cost is high
Solution Approach 1:
The patent segments the DNA sequencing process into minimal essential components: circular template formation through ligation of complementary overhangs and direct sequencing. This segmentation eliminates unnecessary library construction steps, reducing reagent costs and operational complexity while maintaining sequencing accuracy.
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
This method allows for high-accuracy, rapid, and cost-effective detection of both single-stranded and double-stranded DNA molecules, including cell-free DNA, with the ability to use probes as primers for circular sequencing, reducing reaction time and costs.
Implementation Method 1
The partially double stranded linear DNA molecule is incubated with a ligase capable of intra-molecular ligation of single stranded DNA molecules to generate a partially double stranded circular DNA molecule
Implementation Method 2
the method of preparing the partially double stranded linear DNA molecule includes providing a single stranded linear DNA molecule and hybridizing a probe to the single stranded linear DNA molecule
Implementation Method 3
after hybridizing the probe to the single stranded linear DNA molecule, the partially double stranded linear DNA molecule is extracted by a streptavidin bead
Data Source
AI summary
A method of constructing a circular template includes preparing a partially double stranded linear DNA molecule, and incubating the partially double stranded linear DNA molecule with a ligase capable of intra-molecular ligation of single stranded DNA molecules to generate a partially double stranded circular DNA molecule. A method of detecting DNA molecules includes the following steps. Target DNA molecules are isolated with probes to form partially double stranded linear DNA molecules. The partially double stranded linear DNA molecules are incubated with ligases capable of intra-molecular ligation of single stranded DNA molecules to generate partially double stranded circular DNA molecules. A circular sequencing of the partially double stranded circular DNA molecules is conducted by using the probes as primers.