TUMI-Directed DNA Target Detection in Complex Plant Genomes
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Solution Overview
Problem
The complexity and size of plant genomes make direct sequencing for detecting transgenic traits and other nucleotide regions of interest costly and inaccurate, necessitating a more targeted approach for high accuracy and efficiency.
Innovation Solution
The use of targeted unique molecular index (TUMI) sequences in combination with sample index sequences to pool multiple TUMI-containing amplicons for sequencing, where the sequencing primer binding site is oriented away from the targeted nucleotide regions of interest, allowing for directional sequencing of the TUMI without sequencing the target region.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct sequencing is used to detect transgenic traits and nucleotide regions of interest, then comprehensive sequence information can be obtained, but the cost increases and accuracy decreases due to the size and complexity of plant genomes
Solution Approach 1:
The patent extracts and sequences only the TUMI portion of the amplicon by orienting the sequencing primer binding site away from the targeted nucleotide region of interest. This selective extraction of the TUMI sequence eliminates the need to sequence the entire amplicon including the targeted region, thereby reducing cost while maintaining detection accuracy through unique TUMI identification.
2Productivity
If direct sequencing of targeted nucleotide regions is performed, then complete sequence information is obtained, but the sequencing process becomes slower and less efficient
Solution Approach 1:
The patent extracts only the essential TUMI sequence information needed for identification, excluding the targeted nucleotide region of interest from sequencing. This extraction approach accelerates sequencing speed while preserving all necessary information for detecting transgenic traits and nucleotide regions of interest through TUMI-to-target association.
Solution Approach 2:
The TUMI sequence is incorporated into the PCR primer strand before amplification, serving as a pre-prepared identifier. This preliminary inclusion of the TUMI allows for rapid sequencing of the identifier without requiring subsequent sequencing of the targeted region, thereby improving productivity while maintaining detection capability.
3Measurement precision
If the sequencing primer binding site is oriented toward the targeted nucleotide region of interest, then the target region can be sequenced, but the sequencing process becomes less accurate and more time-consuming
Solution Approach 1:
The patent inverts the conventional orientation approach by positioning the sequencing primer binding site to face away from the targeted nucleotide region of interest and toward the TUMI sequence. This inversion allows sequencing to proceed in the direction of the TUMI rather than the target region, improving accuracy by avoiding complex genomic regions while reducing sequencing time through shorter read lengths.
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 provides faster, more accurate, and efficient detection of transgenic traits and other nucleotide regions, enabling universal detection across the agricultural supply chain and enhancing sensitivity to low-level sequences.
Implementation Method 1
initiate sequencing of the amplicon in a direction away from the targeted nucleotide regions of interest
Implementation Method 2
hybridizing a portion of a first PCR primer strand to all or part of a targeted nucleotide region of interest
Implementation Method 3
performing PCR to create one or more amplicons comprising the sequencing primer binding site, the molecular sequence uniquely associated with the targeted nucleotide region of interest
Data Source
AI summary
The disclosed embodiments concern methods for determining sequences of interest using targeted unique molecular index (TUMI) sequences that are uniquely associated with individual polynucleotide fragments in plants, such as those present in a transgenic event, a site-specific mutation or a wild type variant. System, apparatus, and computer program products are also provided for determining a sequence of interest implementing the methods disclosed.