SNP-dependent end labeling for high-throughput genomic analysis
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Solution Overview
Problem
Current methods for determining single nucleotide polymorphisms (SNPs) and copy number variants in genomic DNA lack high-throughput capabilities, hindering research and clinical diagnostics, especially in cytogenetics and cancer studies.
Innovation Solution
A method involving contacting genomic DNA with a restriction endonuclease that cleaves at SNP sites, creating a 5' overhang, followed by extension with labeled nucleotides and hybridization with surface-tethered probes to identify specific alleles, enabling allele-specific analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current SNP determination methods are used, then SNP alleles can be determined, but high-throughput capability is lacking
Solution Approach 1:
The method segments the SNP analysis process into distinct functional modules: restriction enzyme digestion to create allele-specific overhangs, extension enzyme-mediated labeling with fluorescent nucleotides, and hybridization to surface-tethered probes. This modular segmentation enables parallel processing of multiple SNPs simultaneously, achieving high-throughput capability while maintaining manageable complexity through standardized protocol steps.
Solution Approach 2:
The patent introduces intermediary elements including restriction enzymes that recognize SNP-containing sequences and create diagnostic overhangs, extension enzymes that incorporate allele-specific labeled nucleotides, and surface-tethered probes that mediate hybridization. These intermediaries translate genetic information into detectable signals, enabling high-throughput analysis through automated detection systems.
2Measurement precision
If SNP analysis is performed to improve disease mechanism understanding, then diagnostic capability is enhanced, but analysis time increases
Solution Approach 1:
The method performs preliminary actions by using restriction enzymes to pre-process genomic DNA and create allele-specific 5' overhangs before the main detection step. This preliminary digestion step prepares the samples in advance with diagnostic features that enable rapid subsequent detection, reducing overall analysis time while maintaining accurate SNP allele determination.
Solution Approach 2:
The patent replaces traditional mechanical or manual SNP analysis methods with enzymatic and molecular recognition systems. Restriction enzymes and extension enzymes perform specific biochemical transformations, and fluorescent labeling with automated detection substitutes manual analysis, thereby maintaining high measurement precision while significantly reducing analysis time.
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 allows for high-throughput, accurate determination of SNP alleles and copy number variants, enhancing research and diagnostic capabilities in genetics and cancer studies.
Implementation Method 1
contacting a genomic sample comprising double-stranded genomic DNA with a first restriction endonuclease that recognizes a nucleotide sequence that comprises a single nucleotide polymorphism (SNP) site in the double stranded genomic DNA
Implementation Method 2
contacting the digested genomic sample with a extension enzyme and a first labeled nucleotide that is incorporated by the extension enzyme to fill in the overhang only if the overhang comprises a first allele of the SNP
Implementation Method 3
hybridizing the labeled sample with a surface-tethered probe sequence that hybridizes to a nucleic acid fragment that comprises the SNP site
Data Source
AI summary
Certain embodiments described in this disclosure relate to a method of sample analysis. In certain cases, the method comprises: a) contacting a genomic sample comprising double-stranded genomic DNA with a first restriction endonuclease that recognizes a nucleotide sequence that comprises a SNP site in the double stranded genomic DNA, wherein: i. the restriction endonuclease cleaves the genomic DNA at the sequence regardless of the allele of the SNP present at the SNP site; and ii. cleavage of the sequence by the restriction enzyme creates a 5′ overhang that comprises the SNP site; b) contacting the digested genomic sample with a extension enzyme and a first labeled nucleotide that is used by the extension enzyme to fill in the overhang only if the overhang comprises a first allele of the SNP.