AFM DNA Mapping with CRISPR-Cas9 Labeling
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Solution Overview
Problem
Current DNA mapping technologies are expensive, incomplete, computationally intense, or complex, and struggle to accurately resolve long-range genetic variations such as deletions, duplications, inversions, and translocations, particularly in complex diseases like cancer, while existing optical mapping methods are lengthy and require sophisticated protocols.
Innovation Solution
An atomic force microscopy (AFM) system with a scanning probe arrangement and CRISPR-Cas9 labeling technique for high-speed, high-resolution DNA mapping, utilizing a magnesium-free mixture to form a CRISPR-Cas9/DNA complex, which is then imaged on a flat surface to generate precise DNA maps without cleavage, combined with a cost-effective optical pickup unit for displacement sensing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional optical mapping methods are used, then measurement precision is improved, but device complexity and protocol complexity increase
Solution Approach 1:
The patent replaces complex optical mapping systems with atomic force microscopy (AFM), which uses mechanical scanning probes to physically scan and map DNA molecules. This substitution simplifies the overall system by using direct mechanical detection rather than complex optical pathways, while maintaining high measurement precision for DNA structural variations
Solution Approach 2:
The patent extracts and eliminates the need for sophisticated Bayesian procedures and multiple hyper-parameters from the mapping protocol. By using direct AFM imaging of CRISPR-Cas9 labeled DNA, the complex computational and procedural elements of traditional optical mapping are removed, resulting in a simpler, more straightforward protocol
2Measurement precision
If high-resolution DNA mapping is achieved, then measurement precision is improved, but loss of time increases
Solution Approach 1:
The patent performs preliminary labeling of DNA molecules with CRISPR-Cas9 complexes before the AFM imaging step. This pre-labeling approach allows the imaging process to directly detect pre-positioned markers rather than requiring real-time identification, significantly reducing the time required for high-resolution mapping while maintaining precision
3Measurement precision
If conventional AFM systems are used, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The patent uses an optical pickup unit, originally designed for reading optical discs, to detect cantilever displacement in the AFM system. This copying/adaptation approach allows the use of a commercially available, relatively simple device to perform the function of a complex displacement sensor, reducing system complexity and cost while maintaining the precision needed for DNA mapping
Solution Approach 2:
The patent employs an optical pickup unit, a relatively inexpensive and simple device compared to specialized AFM displacement sensors. This choice prioritizes cost-effectiveness and simplicity over using expensive, highly sophisticated detection equipment, making the high-resolution DNA mapping system more accessible and easier to implement
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 system provides high-resolution, cost-effective, and scalable DNA mapping capable of resolving variations from tens to hundreds of thousands of base pairs, facilitating accurate structural genomic variation detection and de novo sequence assembly, complementing sequencing approaches.
Implementation Method 1
incubating a target nucleotide in a magnesium-free mixture, where the mixture includes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-cellular apoptosis susceptibility (Cas) protein and a guide RNA, where incubating the target nucleotide with the CRISPR-Cas protein binds the CRISPR-Cas protein to the target nucleotide to form a CRISPR-Cas/target nucleotide complex without the CRISPR-Cas protein cleaving the target nucleotide
Implementation Method 2
a cantilever(s), a scanning probe arrangement(s) including a laser positioned over a portion of the cantilever(s) which contacts a surface of a sample(s)
Data Source
AI summary
Exemplary embodiments of the present disclosure can include, for example, an atomic force microscopy (AFM) system, including a cantilever(s), an optical pickup unit(s) (OPU(s)) including a laser positioned over the cantilever(s), and a power source providing noise with a noise level that is below 300 Picometers. The noise level of the power source can be below 200 Picometers. A digitizing arrangement can be included which can be associated with the OPU. The digitizing arrangement(s) can have a bandwidth of about 2 MHZ. The OPU(s) can have a detection bandwidth of at least 80 MHZ. The exemplary apparatus can be combined with a chemical protocol and statistical signal processing and image analysis procedures to map DNA at high speed and accuracy.