Multiplex PCR Primer Design Prioritization for Single-Cell Genotyping
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Solution Overview
Problem
Direct multiplex PCR for minute DNA samples with low redundancy, such as genomic DNA from a single cell, imposes restrictive primer design conditions, leading to unsuccessful amplification of candidate amplification regions and reduced accuracy in genotyping analysis.
Innovation Solution
A method for designing primers for multiplex PCR that involves assigning priorities to candidate amplification regions based on their coordinate values, the number of candidate primers available, and variant frequencies, to ensure successful amplification and high accuracy in genotyping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If direct multiplex PCR is performed for minute DNA samples with low redundancy, then the reading capacity of sequencers is充分利用, but the restrictive primer design conditions lead to unsuccessful amplification of candidate amplification regions
Solution Approach 1:
The patent applies preliminary action by pre-evaluating and filtering candidate primers based on multiple parameters (melting temperature, GC content, base sequence length, specificity, and hairpin structure formation) before the actual multiplex PCR reaction. This preliminary selection process ensures that only primers with high compatibility and amplification potential are used, thereby preventing amplification failures while maintaining high productivity.
Solution Approach 2:
The patent employs parameter changes by systematically adjusting and optimizing multiple primer parameters simultaneously, including melting temperature (Tm) within a specific range (55-65°C), GC content (40-60%), and base sequence length (18-24 nucleotides). These parameter optimizations resolve the contradiction by creating primers that are both highly specific and robust for amplifying minute DNA samples.
2Reliability
If restrictive primer design conditions are imposed to ensure amplification success, then amplification reliability improves, but the number of candidate amplification regions that can be successfully amplified decreases
Solution Approach 1:
The patent applies universality by designing a comprehensive primer evaluation system that simultaneously assesses multiple parameters (specificity, Tm, GC content, length, and hairpin formation) using a unified scoring mechanism. This multi-functional evaluation approach allows the system to identify primers that satisfy all design conditions, thereby maintaining high amplification success rates while maximizing the number of amplifiable regions.
Solution Approach 2:
The patent uses copying by creating a virtual model of primer performance through in silico evaluation and scoring. Instead of directly testing all possible primer combinations experimentally, the system copies the evaluation process into a computational framework that predicts amplification success, thereby identifying more successful primer candidates without proportionally increasing experimental workload.
3Reliability
If the number of candidate amplification regions is reduced to ensure successful amplification, then amplification reliability improves, but the accuracy of genotyping analysis deteriorates
Solution Approach 1:
The patent applies preliminary action by pre-selecting and prioritizing candidate amplification regions based on their amplification success probability before conducting the actual genotyping analysis. This preliminary prioritization ensures that the most reliable regions are analyzed first, maintaining high genotyping accuracy while minimizing the impact of any amplification failures.
Solution Approach 2:
The patent employs local quality by applying different evaluation criteria and weighting to different candidate amplification regions based on their individual characteristics. Regions with higher amplification potential are given higher priority and more stringent evaluation, while regions with lower potential are evaluated with adjusted criteria, thereby optimizing the overall set of amplifiable regions for accurate genotyping.
Data Source
AI summary
There is provided a method for designing primers for multiplex PCR, for assigning priorities to candidate amplification regions on same chromosomal DNA and designing primers for PCR amplifying the candidate amplification regions according to the priorities, the method having a feature in a method for assigning priorities.