Nicking Endonuclease DNA Mapping via Flap Formation
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Solution Overview
Problem
Current DNA mapping and sequencing technologies are time-consuming and expensive, particularly for human genomes, due to limitations in read lengths and the inability to accurately assemble sequences with repeats, necessitating improved methods for rapid and cost-effective analysis.
Innovation Solution
The method involves preparing target analyte samples by using nicking endonucleases to create nicks in double-stranded DNA templates, followed by base extension reactions to form single-stranded flaps, which are then coated with binding moieties to enhance electrical detection, allowing for accurate mapping and sequencing using nanopore or fluidic channel systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If short-read sequencing technologies are used, then sequencing cost and time are reduced, but assembly accuracy deteriorates due to inability to correctly assemble sequences with repeats
Solution Approach 1:
The DNA molecule is segmented into distinct regions by introducing nicks at specific locations using nicking endonucleases. Base extension reactions then create flaps that physically segment the DNA, allowing short-read sequences to be accurately mapped to specific genomic locations and assembled correctly even in repetitive regions.
Solution Approach 2:
Nicking endonucleases and base extension reactions serve as intermediary mechanisms that create physical markers (flaps) on the DNA template. These intermediaries enable short-read sequences to be anchored at specific locations, solving the assembly ambiguity problem without requiring long-read sequencing technology.
2Manufacturing precision
If more sequencing data is collected to improve assembly accuracy, then time and cost increase significantly
Solution Approach 1:
Nicking endonucleases perform preliminary action by creating nicks at specific recognition sites before sequencing. Base extension reactions then create flaps that serve as predetermined markers, allowing subsequent short-read sequencing to be efficiently assembled without requiring excessive sequencing depth or time.
3Manufacturing precision
If traditional mapping methods are used, then assembly guidance is provided, but map density and resolution are insufficient for accurate short-read assembly
Solution Approach 1:
The method changes the physical parameters of the DNA template by creating flaps through base extension reactions. This physical modification increases map density and resolution without requiring complex mapping algorithms or multiple mapping steps, as the flaps themselves serve as high-resolution markers.
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 approach enables efficient and accurate nucleic acid mapping and sequencing, improving the assembly of human genomes by differentiating between double-stranded and single-stranded regions, thereby reducing the time and cost of DNA analysis.
Implementation Method 1
contacting the double-stranded DNA template with a nicking endonuclease to form a nick at a sequence-specific nicking location on the first DNA strand
Implementation Method 2
conducting a base extension reaction on the first DNA strand along the corresponding region of the second DNA strand, the reaction starting at the nick and progressing toward the 3' end of the first DNA strand to thereby form a single-stranded flap
Implementation Method 3
coating the double-stranded DNA template and the single-stranded flap with a binding moiety that enhances electrical detection of the template
Implementation Method 4
The application of a constant DC voltage between the two reservoirs results in a baseline ionic current that is measured. If an analyte is introduced into a reservoir, it may pass through the fluidic channel and change the observed current
Data Source
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AI summary
Assay methods and apparatus for the analysis of biopolymers are disclosed. The assays employ nicking endonucleases to enable the generation of flaps on target biomolecules which are detected in nanopore or fluidic channel devices. Identification of flap locations enables a map of the target biomolecule to be derived.