Micropatterned Substrate Linearizes DNA for Optical Mapping
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Solution Overview
Problem
Current methods for DNA mapping, such as restriction mapping, are labor-intensive and require large sample amounts, and they do not provide an ordered sequence of restriction sites, limiting their efficiency in genome assembly.
Innovation Solution
A method involving a micropatterned substrate with alternating binding and non-binding regions is used to immobilize and linearize oligonucleotides. This method involves contacting the substrate with a solution containing oligonucleotides, where one end attaches to the binding region and the molecule extends into the non-binding region, allowing for optical mapping and sequencing library generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional restriction mapping is used, then physical mapping of genome fragments can be achieved, but the process is highly labor-intensive and requires large amounts of sample
Solution Approach 1:
The patent replaces traditional mechanical restriction enzyme cutting and gel electrophoresis with a microfluidic system that uses controlled fluid flow to linearize DNA molecules and position them on a substrate. This substitution of mechanical/biochemical processes with a microfluidic platform dramatically reduces labor intensity and sample requirements while maintaining mapping accuracy.
Solution Approach 2:
The invention changes the physical state and positioning parameters of DNA molecules by using microfluidic flow to stretch and immobilize them in a linear configuration on a solid substrate. This parameter change from solution-phase to surface-immobilized linear configuration enables high-throughput optical mapping with minimal sample input.
2Loss of information
If traditional restriction mapping is used, then a fingerprint of genomic DNA can be obtained, but an ordered sequence of restriction sites is not provided
Solution Approach 1:
The patent replaces traditional restriction enzyme digestion with a microfluidic linearization approach that preserves the complete ordered sequence of restriction sites. By physically stretching and immobilizing intact DNA molecules in linear form, the system maintains all sequence information in order, enabling direct optical mapping without the information loss inherent in traditional fingerprinting methods.
3Length of moving object
If commercial devices are used to stretch single DNA molecules, then DNA linearization can be achieved, but the length of linearized DNA is still short for efficient large-scale genome assembly
Solution Approach 1:
The patent employs a microfluidic channel array substrate that segments the DNA stretching process into controlled flow paths. This segmentation allows multiple DNA molecules to be linearized simultaneously in parallel channels, achieving both sufficient individual molecule length and high-throughput processing for efficient genome assembly.
Solution Approach 2:
The invention transitions from single-molecule manipulation in three-dimensional solution space to two-dimensional surface immobilization in a microfluidic plane. This dimensional change allows long DNA molecules to be stretched and positioned along defined paths on the substrate, maintaining full length while enabling high-density parallel processing for scalable genome assembly.
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 effectively immobilizes and linearizes DNA molecules, enabling efficient optical mapping and DNA sequencing library generation, which addresses the limitations of traditional DNA mapping techniques.
Implementation Method 1
one end of at least one oligonucleotide molecule attaches to the binding region of the micropatterned substrate
Implementation Method 2
combing the at least one oligonucleotide molecule such that the at least one oligonucleotide molecule extends from the binding region into at least a portion of an adjacent non-binding region
Data Source
AI summary
The present invention provides novel methods for immobilizing and/or optically mapping oligonucleotides, the method comprising immobilizing the oligonucleotides on a micropatterned substrate. In another aspect, the invention provides a method for mapping a genome, wherein the method is capable of resolving a single nucleotide polymorphism (SNP), the method comprising introducing to the genome a CRISPR/Cas9 system comprising at least one single-guide RNA (sgRNA) specific for a target sequence or a plurality of target sequences across the genome and a Cas9 D10A, wherein the CRISPR/Cas9 system nick labels the target sequence, and the target sequence or genome is analyzed.