Multiplex Sequencing Library for Endometrial Cancer Gene Mutation Detection
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Solution Overview
Problem
Current methods for detecting endometrial cancer-related gene mutations, such as Sanger sequencing, require a large sample size, significant operator workload, and high costs due to the need for separate amplification and sequencing of long genetic sequences, making them inefficient and costly.
Innovation Solution
A library construction method for high-throughput sequencing that uses specifically designed modified degenerate primers to detect mutations in eight endometrial cancer-related genes (MSH2, PMS2, MLH1, MSH6, EPCAM, TP53, POLE, and PTEN) in a single tube, covering coding regions and exon-intron junctions, requiring only 10 ng of DNA sample and completing library construction in 3.5 hours.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Sanger sequencing method is used to detect eight endometrial cancer-related genes, then detection accuracy is maintained, but detection time increases and productivity decreases
Solution Approach 1:
The patent segments the detection process by designing gene-specific primers for each of the eight target genes (MSH2, PMS2, MLH1, MSH6, EPCAM, TP53, POLE, PTEN), allowing parallel amplification and sequencing of multiple genes simultaneously rather than sequential detection, thereby maintaining accuracy while improving throughput
Solution Approach 2:
The patent creates a universal detection system that can simultaneously detect mutations across all eight endometrial cancer-related genes using a single sequencing run, making the method multi-functional for comprehensive genetic screening without requiring separate experiments for each gene
2Reliability
If separate amplification tubes are used for each gene, then detection reliability is improved, but device complexity and operator workload increase
Solution Approach 1:
The patent merges the amplification of eight different genes into a single multiplex PCR reaction by designing a set of gene-specific primers that can all amplify their respective targets in the same reaction tube, reducing the number of tubes from 200+ to just one while maintaining reliable detection through optimized primer concentrations and annealing conditions
3Manufacturing precision
If long genetic sequences are amplified separately, then sequencing precision is maintained, but loss of time and operational complexity increase
Solution Approach 1:
The patent performs preliminary action by designing and optimizing a comprehensive set of gene-specific primers before the actual detection, allowing all eight genes to be amplified in a single multiplex PCR reaction. This preliminary preparation enables simultaneous sequencing of all targets, reducing the time required compared to sequential amplification while maintaining sequencing precision through optimized primer design
4Quantity of substance
If multiple amplification reactions are performed, then detection completeness is improved, but cost per sample increases
Solution Approach 1:
The patent combines the sequencing of all eight genes into a single high-throughput sequencing run, merging what would otherwise require multiple separate sequencing reactions. This consolidation maintains complete detection of all target genes while significantly reducing the cost per sample by eliminating redundant reagents, equipment usage, and operational overhead associated with multiple separate experiments
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 significantly reduces detection time and cost while enabling efficient, simultaneous detection of multiple genes, facilitating genetic risk assessment and guiding drug treatment.
Implementation Method 1
designing the primers of these genes, and then sequencing the gene sequence in different sections to analyze whether there are mutations
Data Source
AI summary
The present disclosure discloses a library construction method for detecting endometrial cancer-related gene mutations based on high-throughput sequencing, and belongs to the field of biotechnology. The method can detect the mutation types of endometrial cancer-related genes MSH2, PMS2, MLH1, MSH6 EPCAM, TP53, POLE, and PTEN in surgically removed fresh pathological tissues, formaldehyde-fixed and paraffin-embedded pathological tissues, paraffin sections, and specimens of whole blood, plasma, serum, and pleural effusion, etc. It may be used for multiple target sequences in a single tube to quickly complete the library construction. The entire library construction process only takes 3 hours, and the manual operation only needs 30 minutes. Combined with high-throughput sequencing, the platform may effectively solve the current difficulty in the detection of somatic multi-gene all-exon mutations in clinical endometrial cancer samples based on small numbers of clinical samples, and the cost is low.