Single-Cell mtDNA Mutation Detection Using Segmented PCR
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Solution Overview
Problem
Current technology lacks the ability to accurately analyze mitochondrial DNA (mtDNA) mutations in a single egg cell due to the presence of similar sequences in the nucleus and the limited number of egg cells available, making it difficult to extract and analyze mtDNA mutations effectively.
Innovation Solution
A method involving PCR amplification of specific mtDNA regions, followed by nucleic acid sequencing, to detect mtDNA mutations in a single egg cell, using primers with specific nucleotide sequences (SEQ ID NOs: 1-8) and a lysis solution of H2O, 10×KOD buffer, and proteinase K, with controlled temperature and time parameters for lysis and PCR amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification is performed on mtDNA sequences in a single egg cell, then mtDNA mutations can be detected, but mitochondrial nuclear pseudogenes with similar sequences interfere with the detection accuracy
Solution Approach 1:
The full-length mtDNA is divided into multiple predetermined regions that are amplified separately through PCR. This segmentation allows for more precise control over the amplification process and enables the use of region-specific primers that can distinguish mtDNA from nuclear pseudogenes, thereby improving detection accuracy while minimizing interference.
Solution Approach 2:
The patent introduces specific primers as intermediaries that selectively bind to mtDNA sequences rather than nuclear pseudogenes. These primers act as mediators that facilitate the specific amplification of mtDNA regions while excluding the amplification of similar but incorrect sequences, thus resolving the interference problem.
2Quantity of substance
If genome extraction is performed from a small number of egg cells, then mtDNA analysis becomes possible, but insufficient cell numbers make genome extraction difficult
Solution Approach 1:
Instead of attempting to extract and amplify the entire mtDNA genome from trace cells, the patent segments the genome into multiple smaller predetermined regions. This segmentation reduces the amount of DNA required for each PCR reaction, making it feasible to obtain sufficient amplifiable material from just a few egg cells.
Solution Approach 2:
The patent performs partial amplification by selecting and amplifying only specific predetermined regions of the mtDNA rather than the entire genome. This partial action approach requires less starting material and enables successful analysis from trace cell numbers that would be insufficient for complete genome extraction and analysis.
3Measurement precision
If multiple predetermined regions of mtDNA are amplified separately, then full-length mtDNA coverage is achieved, but the detection process becomes more complex
Solution Approach 1:
The patent systematically divides the full-length mtDNA into multiple predetermined regions with defined boundaries. This structured segmentation provides a clear framework for comprehensive coverage while organizing the complexity into manageable, standardized segments that can be processed through a systematic workflow.
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 method achieves high accuracy and sensitivity in detecting mtDNA mutations in a single egg cell, covering the full-length mtDNA and providing insights into mtDNA mutations, which are crucial for understanding infertility and fertility decline.
Implementation Method 1
a lysis solution of H2O, 10×KOD buffer, and proteinase K
Implementation Method 2
subjecting different predetermined regions of the mtDNA to PCR amplification to obtain amplification products
Data Source
AI summary
Provided are a method and a system for detecting mtDNA mutations. Specifically, provided is a method for detecting mtDNA mutations in trace cells, comprising: (a) extracting mtDNA in trace cells, the number of trace cells being 1 to 4; (b) performing PCR amplification on different predetermined regions of the mtDNA to acquire amplification products of the different predetermined regions, the different predetermined regions of the mtDNA being superimposed to form the full-length mtDNA; (c) mixing the amplification products of the different predetermined regions to obtain an mtDNA full-length nucleic acid sequencing library; (d) sequencing the nucleic acid sequencing library to obtain sequencing results; and (e) on the basis of the sequencing results, determining mtDNA mutations in the trace cells to be tested.