SE33 Primer Set Design for Genotype Concordance
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Solution Overview
Problem
The SE33 locus in DNA typing experiences issues with allelic dropout and mobility shifts due to single nucleotide polymorphisms (SNPs) in the primer-binding sites, leading to discordant results in DNA profiling, particularly affecting the accuracy of DNA-based technologies in forensic and diagnostic applications.
Innovation Solution
A nucleic acid composition and primer set design that incorporates SNPs at specific positions within the SE33 locus, including variants at positions 316, 317, and 324, to stabilize primer-binding sites and prevent allelic dropout, utilizing nucleotide analogs and universal bases to ensure accurate amplification and detection by capillary electrophoresis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard PCR amplification is used for SE33 locus, then DNA profiling can be performed, but allelic dropout and mobility shifts occur due to SNPs in primer-binding sites
Solution Approach 1:
The patent applies preliminary action by designing and incorporating SNP-aware primers before the PCR amplification process. The primers are pre-engineered to account for known SNP variants at positions 316, 317, and 324 in the SE33 locus, ensuring that amplification will succeed regardless of which SNP variant is present in the sample. This prevents allelic dropout before it can occur.
Solution Approach 2:
The patent applies parameter changes by modifying the primer sequences to include nucleotide analogs and universal bases that can accommodate SNP variations. Specifically, the primers are designed with altered binding parameters at the SNP positions, allowing them to bind effectively to both wild-type and variant sequences, thereby maintaining amplification efficiency across different genetic backgrounds.
2Productivity
If conventional primer design is used, then amplification can proceed, but mobility shifts occur affecting genotype concordance
Solution Approach 1:
The patent applies the intermediary principle by introducing nucleotide analogs and universal bases as mediators in the primer sequences. These intermediaries act as buffer zones that can accommodate the SNP variations without disrupting the overall primer-template binding. The nucleotide analogs serve as flexible intermediaries that maintain binding stability despite the underlying genetic variation, preventing mobility shifts in the amplified products.
Solution Approach 2:
The patent applies local quality by making specific modifications only at the SNP-affected regions of the primers while keeping the rest of the primer sequence standard. The modifications are localized to positions 316, 317, and 324 where SNPs are known to occur, allowing the primers to maintain high specificity and binding affinity at these critical locations while preserving overall amplification efficiency.
3Adaptability or versatility
If SNPs are present in primer-binding sites, then genetic diversity is captured, but allelic dropout reduces detection sensitivity
Solution Approach 1:
The patent applies universality by designing primers that can universally bind to multiple SNP variants at the SE33 locus. The primers incorporate universal bases and nucleotide analogs that allow a single primer sequence to effectively bind to both wild-type and various SNP-containing templates. This multi-functional design ensures that the same primer can detect all genetic variants without requiring separate primers for each variant, thereby maintaining both genetic diversity coverage and detection sensitivity.
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
The solution enhances the specificity and sensitivity of DNA profiling by minimizing allelic dropout and mobility shifts, achieving concordance between different DNA kits and improving the reliability of DNA-based technologies in identifying individuals and determining genetic relationships.
Implementation Method 1
detected by capillary electrophoresis
Data Source
AI summary
Disclosed are primer set compositions, methods and kits for human identification using the highly complex sequence locus, SE33 (ACTBP2) in single and multiplex PCR reactions. Additionally, disclosed are three newly discovered single nucleotide polymorphisms (SNPs) within the SE33 locus that can cause discordance seen as mobility shift or allelic dropout. Also disclosed are kits useful in human identification.