Isolating Large DNA Fragments via Destabilized Region Tagging
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Solution Overview
Problem
Current methods for selectively isolating large DNA fragments are expensive and inefficient, often requiring denaturation steps and resulting in non-specific capture of unwanted DNA fragments, which limits their effectiveness in genomic re-sequencing and genetic defect diagnosis.
Innovation Solution
A method involving the use of single-stranded polynucleotides with linker and capture portions to selectively target and capture target double-stranded polynucleotides with internal destabilized regions, utilizing techniques like nick-translation DNA synthesis and Uracil-DNA glycosylase treatment to create abasic sites, allowing for specific interaction with capture substances without denaturation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If denaturation steps are used to isolate large DNA fragments, then the isolation process can be performed, but the method becomes expensive and results in non-specific capture of unwanted DNA fragments
Solution Approach 1:
The patent introduces a preliminary tagging step where single-stranded polynucleotides are attached to specific regions of the double-stranded target DNA before capture. This preliminary action creates unique identifiers on the target molecules, enabling specific recognition and capture without requiring denaturation, thereby improving reliability while maintaining process simplicity
Solution Approach 2:
The patent uses single-stranded polynucleotides as intermediary molecules that bridge the target double-stranded DNA and the capture beads. These intermediaries carry capture moieties that specifically bind to the target, enabling specific capture without direct interaction between the beads and the target DNA, thus improving reliability without increasing complexity
2Quantity of substance
If current methods are used for selective isolation, then DNA fragments can be isolated, but the cost is high and large DNA fragments cannot be effectively isolated
Solution Approach 1:
The patent employs inexpensive single-stranded polynucleotide tags with capture moieties as disposable intermediaries for capturing large DNA fragments. These tags can be synthesized cost-effectively and enable the isolation of large DNA fragments without requiring expensive equipment or reagents, thereby improving the quantity of substance isolatable while reducing manufacturing cost
Solution Approach 2:
The patent changes the fundamental parameter of how DNA fragments are tagged for capture - instead of using traditional methods that work for small fragments, it employs a tagging system that specifically enables the isolation of large DNA fragments. This parameter change allows effective isolation of larger fragments without proportionally increasing cost, as the same tagging approach scales to different fragment sizes
3Reliability
If traditional capture methods are used, then DNA can be isolated, but non-specific capture of unwanted DNA fragments occurs
Solution Approach 1:
The patent segments the capture system into distinct functional components: a target-specific recognition element (the single-stranded polynucleotide tag hybridized to the target) and a capture element (the bead with capture moiety). This segmentation allows the recognition element to provide high accuracy of target selection through specific hybridization, while the capture element provides high purity through specific binding, resolving the contradiction between accuracy and purity
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
Enables efficient and specific isolation of large DNA fragments, improving genomic re-sequencing and genetic defect diagnosis by reducing costs and minimizing non-specific capture, thereby enhancing the accuracy and reliability of molecular biology tools.
Implementation Method 1
the linker portion having a sequence sufficiently complementary to hybridize to the intact polynucleotide strand of the destabilized region of the target
Implementation Method 2
treating with Uracil-DNA glycosylase (UDG)
Data Source
AI summary
Methods of isolating target double-stranded polynucleotides with internal single-stranded regions are provided. Compositions and kits comprising double-stranded polynucleotides with internal single-stranded regions are also provided.