Universal Blocking Oligos for Multiplexed Nucleic Acid Sequencing
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Solution Overview
Problem
Current nucleic acid manipulation and sequencing methods are costly, time-consuming, and prone to contamination, especially in high-throughput multiplexed capture reactions, due to the need for multiple specific blocking oligonucleotides and inefficient hybridization capture processes.
Innovation Solution
The use of universal blocking nucleic acids (U-block nucleic acids) that do not hybridize to nucleic acid barcode sequences, allowing for efficient multiplex sequencing without the need for multiple specific blocking oligonucleotides, and a modified hybridization capture method that eliminates vacuum and heat-based concentration steps, using magnetic beads and biotinylated DNA baits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple specific blocking oligonucleotides are used for each barcode sequence, then hybridization specificity is improved, but device complexity and cost increase
Solution Approach 1:
The patent applies universality by designing a single set of blocking oligonucleotides that can block multiple different barcode sequences simultaneously. The blocking oligonucleotides are configured to hybridize to universal regions flanking the barcodes rather than to each specific barcode sequence individually, allowing one blocking oligo to serve multiple functions across different samples and barcodes, thereby reducing the total number of blocking oligonucleotides needed while maintaining hybridization specificity
Solution Approach 2:
The patent extracts the barcode sequences from the regions where blocking is needed by designing blocking oligonucleotides that target only the universal adapter regions flanking the barcodes. This extraction approach allows the blocking function to be separated from the barcode-specific sequences, enabling a universal blocking strategy that doesn't require individual blocking oligos for each barcode while still preventing unwanted hybridization
2Productivity
If traditional hybridization capture methods are used, then capture efficiency is improved, but processing time and contamination risk increase
Solution Approach 1:
The patent applies preliminary action by performing blocking oligonucleotide hybridization before the hybridization capture step. This preliminary blocking prevents unwanted hybridization events from occurring during subsequent processing steps, reducing contamination risk and eliminating the need for time-consuming vacuum concentration steps and heat-based processing that are required in traditional methods to manage contamination
Solution Approach 2:
The patent replaces mechanical concentration steps (vacuum drying) and heat-based processing with a biochemical approach using blocking oligonucleotides and magnetic bead-based capture. This substitution eliminates the need for mechanical manipulation that increases contamination risk and reduces processing time by allowing direct aqueous-phase hybridization and magnetic separation without intermediate drying or heating steps
3Quantity of substance
If vacuum and heat-based concentration steps are used, then nucleic acid concentration is improved, but contamination risk increases
Solution Approach 1:
The patent introduces blocking oligonucleotides as intermediaries that bind to unwanted nucleic acid sequences, preventing them from participating in unwanted hybridization events. This intermediary approach allows nucleic acid concentration to be achieved through magnetic bead-based capture in aqueous solution without requiring vacuum concentration steps, thereby maintaining nucleic acid concentration while eliminating the contamination risk associated with vacuum processing and open-heated steps
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 approach reduces contamination, increases efficiency, and lowers costs by enabling automated, high-throughput nucleic acid sequencing with improved data quality and reduced processing time, while maintaining reaction kinetics and component concentrations.
Implementation Method 1
a first and second U-block nucleic acid are configured to hybridize to the first non-native nucleic acid on opposing sides of the first distinguishable nucleic acid barcode and (ii) a third and fourth U-block nucleic acid are configured to hybridize to the second non-native nucleic acid on opposing sides of the second distinguishable nucleic acid barcode
Implementation Method 2
the capture nucleic acids are configured to specifically hybridize to a subset of the one or more library inserts
Data Source
AI summary
Provided herein, in some embodiments, are novel compositions and improved methods for nucleic acid manipulation and analysis that can be applied to multiplex nucleic acid sequencing. In certain embodiments, the novel compositions and methods presented herein are more cost effective, more conducive to automation, and faster than traditional approaches. Also provided herein are novel blocking nucleic acids.