Multiplex Mitochondrial DNA Quantitation With Deletion Detection
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Solution Overview
Problem
Existing methods for quantifying mitochondrial nucleic acid relative to genomic nucleic acid are inefficient and require control samples or internal standards, and do not allow for accurate determination of mitochondrial dosage and detection of deletions in a single reaction.
Innovation Solution
Multiplex methods and kits that amplify sets of mitochondrial and genomic polynucleotides under specific conditions, allowing for direct comparison and determination of relative dosage without the need for control samples, and enable detection of mitochondrial deletions by examining multiple regions of the mitochondrial genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing methods are used to quantify mitochondrial nucleic acid, then control samples or internal standards are required, but this increases device complexity and requires optimization
Solution Approach 1:
The assay uses endogenous nuclear DNA within the same sample to serve as an internal reference for normalizing mitochondrial DNA quantification. This self-service approach eliminates the need for separate control samples or additional internal standards, reducing assay complexity while maintaining measurement precision through within-sample normalization
Solution Approach 2:
The method combines mitochondrial DNA quantification and nuclear DNA normalization into a single multiplex reaction. By merging these functions into one assay, the patent eliminates the need for separate control samples and multiple optimization steps, reducing device complexity while maintaining accurate mitochondrial dosage determination
2Measurement precision
If multiple separate reactions are used for mitochondrial quantification and deletion detection, then measurement precision improves, but productivity decreases
Solution Approach 1:
The patent merges mitochondrial DNA quantification, nuclear DNA normalization, and deletion detection into a single multiplex reaction. This allows simultaneous measurement of multiple parameters in one assay, improving productivity by eliminating the need for multiple separate reactions while maintaining measurement precision through within-sample comparisons
Solution Approach 2:
The multiplex assay performs multiple functions simultaneously: quantifying mitochondrial DNA dosage, detecting mitochondrial deletions, and normalizing to nuclear DNA content. This multi-functionality allows a single reaction to provide comprehensive mitochondrial analysis, improving throughput without sacrificing measurement precision
3Measurement precision
If control samples are required for normalization, then measurement precision improves, but loss of time increases
Solution Approach 1:
The assay uses endogenous nuclear DNA within the same sample to serve as an internal reference for normalization. This eliminates the need for separate control samples, reducing assay time while maintaining measurement precision through within-sample normalization that accounts for variations in DNA extraction and amplification efficiency
4Measurement precision
If internal standards are used, then measurement precision improves, but device complexity and loss of substance increase
Solution Approach 1:
The method uses endogenous nuclear DNA within the sample itself as the normalization reference, eliminating the need for added internal standards. This reduces device complexity by removing the need for standard preparation and addition steps, reduces loss of substance by avoiding dilution with standard solutions, while maintaining quantification accuracy through within-sample normalization
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
Enables accurate assessment of mitochondrial dosage and detection of deletions in a single reaction, minimizing technical variability and requiring no optimization or internal standards, while allowing for the determination of cell state, disease presence, or therapeutic response.
Implementation Method 1
hybridizing the amplification primer pair to mitochondrial and nuclear polynucleotides under hybridization conditions
Implementation Method 2
subjecting the hybridized primer to polymerization conditions
Data Source
AI summary
Provided herein are products and processes for the quantitation of mitochondrial nucleic acid in a sample from a subject. In certain aspects are multiplex methods for determining dosage of mitochondrial nucleic acid relative to genomic nucleic acid for a sample from a subject including amplifying sets of mitochondrial polynucleotides and genomic polynucleotides from nucleic acid for a sample under amplification conditions. In certain aspects are multiplex methods for determining dosage of mitochondrial nucleic acid relative to genomic nucleic acid for a sample from a subject including amplifying sets of mitochondrial polynucleotides and amplifying sets of nuclear polynucleotides from nucleic acid for a sample under amplification conditions.