Maize Pericarp Genotyping via Cell Dissociation and CTAB Buffer
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Solution Overview
Problem
Current methods for pericarp removal from seeds are inefficient, often requiring chemicals that can damage DNA, are labor-intensive, and fail to achieve pure maternal lineage analysis due to contamination from interior seed tissues, necessitating a method for accurate and efficient isolation and purification of pericarp tissue for molecular analysis.
Innovation Solution
A method involving soaking single maize seeds in water, followed by washing and dissociating the pericarp tissue using a cell dissociator, centrifugation to obtain a supernatant for molecular analysis, and optionally using whole genome amplification to achieve sufficient DNA yield, allowing for accurate maternal lineage determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical solutions (NaOH, H2O2) are used for pericarp removal, then pericarp loosening and separation efficiency are improved, but DNA damage and contamination from interior seed tissue occur
Solution Approach 1:
The patent changes the chemical parameter from harsh chemicals (NaOH, H2O2) to a milder chemical composition (CTAB buffer with specific pH and ionic strength), and adjusts the time parameter from prolonged soaking to a optimized time frame, thereby achieving pericarp separation without DNA damage
Solution Approach 2:
The patent introduces CTAB (cetyltrimethylammonium bromide) as an intermediary substance that facilitates pericarp separation while being less harmful to DNA compared to traditional chemicals. The CTAB buffer acts as a mediator between the pericarp tissue and the separation process
2Ease of operation
If chemical soaking is used for pericarp removal, then pericarp loosening is improved, but processing time and chemical handling costs increase
Solution Approach 1:
The patent optimizes the soaking time parameter and chemical composition parameters to achieve effective pericarp loosening in a reduced time frame, balancing ease of operation with time efficiency
3Object-affected harmful factors
If water soaking is used for pericarp removal, then DNA damage is avoided, but pericarp loosening effectiveness and separation purity decrease
Solution Approach 1:
The patent introduces CTAB buffer as an intermediary that provides the chemical assistance needed for effective pericarp separation while maintaining DNA integrity, bridging the gap between gentle water soaking and harsh chemical treatment
Solution Approach 2:
The patent modifies the soaking solution from pure water to a specifically formulated CTAB buffer with controlled pH and ionic strength, enabling effective separation without the harsh effects of traditional chemicals
4Quantity of substance
If multiple seeds are pooled for DNA analysis, then sufficient DNA yield is achieved, but contamination risk and labor requirements increase
Solution Approach 1:
The patent extracts and analyzes DNA from individual pericarp tissues separately rather than pooling multiple seeds, eliminating cross-contamination risks while maintaining sufficient DNA yield through optimized extraction and amplification protocols
5Manufacturing precision
If manual pericarp removal is used, then DNA purity is maintained, but labor time and processing efficiency decrease
Solution Approach 1:
The patent uses CTAB buffer as a chemical intermediary that facilitates automated or semi-automated pericarp separation, combining the purity benefits of careful manual removal with the efficiency of chemical-assisted separation processes
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 the isolation of pure maternal DNA from a single seed, reducing contamination and labor, and achieving high-quality genetic analysis with sufficient DNA yield for accurate maternal lineage determination.
Implementation Method 1
dissociating and homogenizing the pericarp tissue in water or Tris-EDTA buffer using a cell dissociator to obtain a homogenized solution
Implementation Method 2
centrifuging the homogenized solution obtained in step (d) to obtain supernatant
Data Source
Figure 1a~1c
Figure 2a~2b
Figure 3
AI summary
The invention includes methods for obtaining samples of maternal tissue from seeds, obtaining genetic material from the maternal seed tissue, and performing a molecular analysis on the genetic material from the maternal seed tissue to determine maternal lineage of a single seed. The invention also includes methods for establishing a consensus maternal genotype from maternal seed tissues obtained from multiple seeds as well as methods for determining paternal lineage.