Restriction Enzyme DNA Library for SNP Detection
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Solution Overview
Problem
Current methods for detecting single nucleotide polymorphisms (SNPs) are costly, require specialized equipment, and struggle with designing probes for unknown SNPs, as well as introducing deviations in sequencing data due to complex processes and high demand for chip synthesis.
Innovation Solution
A method involving digestion of genomic DNA with restriction endonucleases like Mbo II and Tsp 45I, followed by end-repairing and ligating with sequencing adaptors to create a DNA library, which is then sequenced to obtain SNPs information, reducing costs and improving stability and repeatability of data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If probe-based methods are used for SNP detection, then detection specificity is improved, but probe design complexity and cost increase
Solution Approach 1:
The patent extracts and sequences specific genomic regions containing SNPs by digesting genomic DNA with restriction endonucleases and selecting fragments of specific length ranges. This extracts the relevant SNP-containing sequences from the whole genome, enabling focused analysis without requiring complex probe design for the entire genome.
Solution Approach 2:
The patent creates copies of SNP-containing genomic fragments through PCR amplification after restriction digestion. These amplified copies are then sequenced to detect SNPs, eliminating the need for complex probe-based detection while maintaining detection capability through direct sequencing of the copied fragments.
2Productivity
If commercial SNP chips are used, then large-scale SNP detection capability is improved, but equipment and software requirements increase
Solution Approach 1:
The patent creates a universal DNA library preparation method that can be applied to detect SNPs across the entire genome without requiring specialized SNP-specific equipment. The same restriction digestion and library preparation protocol works for any SNP detection project, making the system universally applicable rather than requiring specialized commercial chip platforms.
Solution Approach 2:
The patent replaces the mechanical and specialized hardware systems of commercial SNP chips with a biochemical approach using restriction endonucleases, PCR amplification, and next-generation sequencing. This substitution eliminates the need for specialized scanning equipment and proprietary software while maintaining large-scale detection capability.
3Loss of information
If RAD sequencing method is used, then SNP discovery capability is improved, but process complexity and deviation introduction increase
Solution Approach 1:
The patent performs preliminary restriction enzyme digestion and fragment selection before sequencing to enrich for SNP-containing regions. By pre-processing the genomic DNA to isolate relevant fragments, the method improves SNP discovery capability while reducing the complexity of the overall process compared to whole-genome RAD sequencing.
Solution Approach 2:
The patent optimizes specific parameters such as fragment length selection (200-500 bp or 300-600 bp ranges) and restriction enzyme choices to improve SNP detection while simplifying the process. These parameter optimizations reduce deviation introduction by standardizing the library preparation protocol and improving sequencing quality without requiring complex process variations.
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 simplifies the process, reduces costs, and enhances the stability and repeatability of sequencing data, enabling efficient SNPs detection and genotyping with improved accuracy and scalability for large-scale samples.
Implementation Method 1
digesting a genomic DNA sample using a restriction endonuclease to obtain a digested product, wherein the restriction endonuclease comprises at least one selected from the group consisting of Mbo II and Tsp 45I
Implementation Method 2
ligating the DNA fragment having the terminal base A with a sequencing adaptor to obtain the DNA library
Data Source
AI summary
A DNA library, and a preparing method thereof, a method of determining DNA sequence information, an apparatus and a kit for detecting SNPs, and a method for genotyping may be provided. The method for preparing the DNA library may comprise the steps of: digesting a genomic DNA sample using a restriction endonuclease to obtain a digested product, wherein the restriction endonuclease comprises at least one selected from the group consisting of Mbo II and Tsp 45I; separating the digested product to obtain DNA fragments having a length of 100 bp to 1,000 bp; end-repairing the DNA fragments to obtain an end-repaired DNA fragments; adding a base A to the end of the end-repaired DNA fragments to obtain DNA fragments having a terminal base A; and ligating the DNA fragments having the terminal base A with sequencing adaptors to obtain the DNA library.