High Throughput Mutation Screening via Sequencing
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Solution Overview
Problem
Current methods for screening mutagenized populations for mutations, such as TILLING, are inefficient and cumbersome, particularly due to reliance on CEL I enzyme which is sensitive to reaction conditions, lacks sensitivity at PCR product termini, and cannot distinguish missense from nonsense mutations, leading to unnecessary screening efforts.
Innovation Solution
High-throughput sequencing strategies are employed to isolate, pool, and amplify genomic DNA from mutagenized populations, followed by sequencing and clustering to identify mutations, eliminating the need for CEL I digestion and gel scoring, thereby enhancing sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CEL I enzyme-based screening is used, then mutation detection can be performed, but the method is sensitive to reaction conditions and lacks reliability
Solution Approach 1:
The patent replaces the CEL I enzyme-based mechanical screening system with a molecular biology-based PCR and sequencing system. Instead of using CEL I enzyme to digest heteroduplex DNA and visualize mutations through gel electrophoresis, the invention uses PCR amplification followed by direct sequencing, eliminating the mechanical/enzymatic digestion step and its associated sensitivity to reaction conditions.
Solution Approach 2:
The patent changes the detection parameters from enzyme activity-based detection (CEL I digestion) to sequence-based detection (direct sequencing). This parameter change transforms the screening method from one sensitive to enzymatic reaction conditions to one that directly reads the DNA sequence, thereby improving reliability and reducing sensitivity to reaction conditions.
2Measurement precision
If CEL I enzyme method is used, then mutation screening is possible, but sensitivity at PCR product termini is insufficient
Solution Approach 1:
The patent replaces the CEL I enzyme digestion mechanism with direct sequencing technology. The sequencing method provides uniform sensitivity across the entire PCR product length, including termini regions, whereas CEL I enzyme showed reduced sensitivity at the ends of PCR products. This substitution eliminates the positional sensitivity variation inherent in the enzyme-based method.
3Productivity
If CEL I enzyme screening is used, then all mutations can be detected, but missense and nonsense mutations cannot be distinguished, leading to unnecessary screening efforts
Solution Approach 1:
The patent changes the detection parameter from binary presence/absence detection (CEL I method that only shows if a mutation exists) to sequence-based detection that provides full sequence information. This allows direct determination of mutation type (missense, nonsense, silent, etc.) from the sequenced data, eliminating the information loss that occurs with CEL I enzyme screening.
Solution Approach 2:
The patent introduces sequence information as an intermediary between mutation detection and functional assessment. Instead of directly assessing all detected mutations for functional impact (as required with CEL I method), the sequenced data serves as an intermediary that allows pre-filtering of mutations by type, reducing the number of mutations that require further functional screening.
4Loss of time
If traditional screening methods are used, then mutation identification can be performed, but the process is cumbersome and time-consuming
Solution Approach 1:
The patent replaces the multi-step mechanical screening process (CEL I digestion, gel electrophoresis, band scoring, confirmation sequencing) with a streamlined PCR and direct sequencing workflow. This substitution eliminates multiple manual操作步骤 and reduces the overall screening time while maintaining or improving detection accuracy.
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 significantly reduces the effort required to identify mutations affecting gene function, allowing for more efficient assessment of mutations and their impact on gene function, while avoiding the limitations of CEL I-based methods.
Implementation Method 1
amplifying the target sequence with a pair of (optionally labeled) primers from the DNA pools
Implementation Method 2
determining the nucleotide sequence of the products and/or fragments using high throughput sequencing
Data Source
AI summary
Efficient methods are disclosed for the high throughput identification of mutations in genes in members of mutagenized populations. The methods comprise DNA isolation, pooling, amplification, creation of libraries, high throughput sequencing of libraries, preferably by sequencing-by-synthesis technologies, identification of mutations and identification of the member of the population carrying the mutation and identification of the mutation.