Tm-Enhanced Oligonucleotides for NGS Target Enrichment
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Solution Overview
Problem
Current nucleic acid hybridization methods for next-generation sequencing face inefficiencies due to off-target nucleic acid interactions, particularly with repetitive DNA elements and terminal adaptor sequences, leading to contamination and reduced capture efficiency of desired sequences.
Innovation Solution
The use of Tm-enhanced oligonucleotides, including locked nucleic acid and bicyclic nucleic acid groups, as blockers and baits to improve target enrichment by enhancing the melting temperature of specific oligonucleotide interactions, thereby reducing off-target selection and increasing the efficiency of nucleic acid selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional hybridization methods are used for nucleic acid selection, then the process is simple and requires fewer modifications, but off-target nucleic acid interactions occur leading to contamination and reduced capture efficiency
Solution Approach 1:
The patent modifies the melting temperature parameter of oligonucleotide probes by incorporating Tm-enhancing groups (such as LNA, BNA, or PNA modifications) to create probes with higher melting temperatures. This parameter change allows the probes to specifically bind to target sequences at lower temperatures, preventing off-target interactions while maintaining high capture efficiency for desired sequences.
Solution Approach 2:
The patent introduces blocking oligonucleotides as intermediary molecules that compete with off-target sequences for binding to the probes. These blockers are designed to bind to repetitive DNA elements and terminal adaptor sequences, preventing them from interacting with the probes and thereby reducing off-target selection without affecting target capture.
2Manufacturing precision
If standard oligonucleotide probes are used, then the method is straightforward and cost-effective, but repetitive DNA elements and terminal adaptor sequences cause contamination in the sequencing library
Solution Approach 1:
The patent converts the harmful effect of repetitive DNA elements and terminal adaptor sequences into a beneficial blocking mechanism. By designing blocking oligonucleotides that specifically recognize and bind to these repetitive elements, the patent transforms what were previously sources of contamination into controlled interactions that actively prevent off-target binding, thereby improving sequencing library quality.
Solution Approach 2:
The patent changes the thermal parameters of the hybridization reaction by using Tm-enhanced probes with higher melting temperatures. This allows the reaction to proceed at lower temperatures where the blocking oligonucleotides can effectively compete with repetitive elements, converting potential contaminants into controlled binding events that improve overall library quality.
3Productivity
If hybrid capture method is used to enrich desired sequences, then fewer enzymatic procedures are required, but off-target interactions still reduce the enrichment efficiency
Solution Approach 1:
The patent modifies the thermal parameters of the hybridization reaction by incorporating Tm-enhancing groups into the probes, creating probes with elevated melting temperatures. This parameter change enables the probes to maintain specific binding to target sequences at lower reaction temperatures, thereby improving both the speed (productivity) and accuracy (reliability) of target enrichment simultaneously.
Solution Approach 2:
The patent introduces blocking oligonucleotides as intermediary molecules that mediate the interaction between probes and off-target sequences. These blockers act as competitive inhibitors that prevent non-specific binding, thereby enhancing the reliability of target enrichment without requiring additional enzymatic steps, thus maintaining high productivity.
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 significantly enhances the enrichment of desired nucleic acid templates relative to undesired ones, improving the quality of sequencing libraries by minimizing off-target sequences and increasing the proportion of target sequences in the sequencing pool.
Implementation Method 1
The use of Tm-enhanced oligonucleotides, including locked nucleic acid and bicyclic nucleic acid groups, as blockers and baits to improve target enrichment by enhancing the melting temperature of specific oligonucleotide interactions
Implementation Method 2
Nucleic acid hybridization has a significant role in biotechnology applications pertaining to identification, selection, and sequencing of nucleic acids
Data Source
AI summary
The invention is directed to modified oligonucleotide compositions and methods for selectively reducing unwanted nucleic acid contaminants and enriching for desired nucleic acid targets from complex genomic nucleic acid mixtures for sequencing applications. The modified oligonucleotide compositions include one or more modified groups that increase the Tm of the resultant oligonucleotide composition.