Mutation Identification Method Using Segmented PCR Primers
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Solution Overview
Problem
Current methods for identifying the exact position of genetic mutations, such as deletions, insertions, and translocations, suffer from low sensitivity and specificity, and are limited in detecting mutations further than a few hundred base pairs from a known sequence, failing to provide precise information on the start or end points of genetic modifications.
Innovation Solution
A method involving PCR with degenerate and specific primers, where the primers hybridize within or outside the ascertained nucleic acid region, allowing for precise amplification and sequencing to determine the mutation's position, using a two-step hybridization process to enhance specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR-based methods (LM-PCR, i-PCR, TAIL-PCR) are used to identify mutation positions, then amplification of DNA fragments can be achieved, but detection sensitivity and specificity remain low and mutations beyond a few hundred base pairs cannot be detected
Solution Approach 1:
The method segments the detection process into two distinct phases: first determining the ANA region boundaries using array-based techniques (CGH array, SNP array, or MLPA), then performing targeted PCR amplification within those defined boundaries. This segmentation allows each phase to optimize for its specific function, resolving the contradiction between detection range and precision.
Solution Approach 2:
The patent applies preliminary action by first determining the ANA region boundaries before performing PCR amplification. Techniques such as CGH array, SNP array, or MLPA are used in advance to identify the precise boundaries of the mutated region, which then guides the subsequent PCR primers design and amplification process, ensuring high sensitivity and specificity from the start.
2Area of stationary object
If array-based techniques (CGH array, SNP array) are used to analyze the whole genome, then comprehensive coverage is achieved, but resolution capacity is limited and deletions or insertions less than 50 Kb cannot be detected
Solution Approach 1:
The methodology segments the detection approach by first using array-based techniques to identify the ANA region boundaries across the genome, then applying high-resolution PCR-based methods specifically within those identified regions. This two-stage segmentation allows comprehensive genome coverage at low resolution followed by targeted high-resolution analysis.
Solution Approach 2:
The ANA region determination acts as an intermediary step between low-resolution array-based screening and high-resolution PCR analysis. This intermediary process identifies precise boundaries that guide subsequent amplification, enabling the system to achieve both broad coverage and high resolution detection capabilities.
3Reliability
If MLPA technique is used to analyze genetic regions, then copy number alterations can be detected, but processivity is very low and only a limited number of genetic regions can be analyzed
Solution Approach 1:
The patent employs MLPA or similar array-based techniques to universally determine ANA region boundaries across the entire genome, regardless of the specific mutation type or location. This universal boundary determination then enables subsequent PCR analysis to be applied to any number of different genetic regions, significantly increasing productivity while maintaining the reliability of copy number alteration detection.
4Ease of manufacture
If conventional PCR methods are used with primers hybridizing far from the mutation site, then amplification can be achieved, but exact mutation limits cannot be identified
Solution Approach 1:
The patent applies preliminary action by first determining the precise ANA region boundaries using array-based techniques before designing and performing PCR amplification. This preliminary boundary identification ensures that PCR primers are positioned optimally close to the mutation site, enabling both successful amplification and precise mutation boundary identification.
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 enables accurate identification and characterization of genetic mutations, including their exact limits, improving detection sensitivity and specificity, and can detect mutations beyond previous limitations, facilitating diagnosis and screening of associated pathologies.
Implementation Method 1
a specific primer which hybridizes specifically: within the ANA region, at a distance of 0.2-100 Kb from any of the ends of the ANA region
Implementation Method 2
a degenerate primer, and where in the PCR reaction the hybridization step comprises two consecutive hybridizations, a first hybridization at a hybridization temperature equal to the Tm of the specific primer ±10° C., and a second hybridization at a hybridization temperature equal to the Tm of the degenerate primer ±5° C.
Implementation Method 3
carrying out a PCR, where the PCR template is DNA extracted from a biological sample taken from the subject under study
Data Source
AI summary
The present invention relates to a method for identifying the position of a genetic mutation and to the use of said method for simplifying the screening of said genetic mutation.