Dual Probe Primer Extension for Variant Allele Enrichment
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Solution Overview
Problem
Current methods for nucleic acid sequencing target enrichment are slow, cumbersome, and costly, particularly when enriching for specific variant alleles, and do not efficiently accommodate unknown structural variations.
Innovation Solution
A method involving unidirectional dual probe primer extension, where oligonucleotides are hybridized and extended with polymerases to form primer extension complexes, captured, and amplified using complementary adapters, allowing for efficient enrichment of target nucleic acids, including variant alleles, with optional solid support binding and modified nucleotides for enhanced capture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If hybridization capture is used to enrich target nucleic acids, then the ability to capture exome-sized regions and regions with unknown structural variations is improved, but the protocol time increases to well over 8 hours and becomes complex
Solution Approach 1:
The capture process is divided into two stages: first, a first oligonucleotide hybridizes to the target and is extended to form a first primer extension complex that is captured and enriched; second, a second oligonucleotide hybridizes to the target and is extended to form a second primer extension complex, releasing the first oligonucleotide. This segmentation allows the patent to achieve comprehensive capture capability while reducing overall protocol time through parallel processing of multiple targets.
Solution Approach 2:
The patent performs preliminary enrichment of primer extension complexes before the final amplification step. By capturing and enriching the first and second primer extension complexes that contain the target nucleic acids, the method prepares the sample for subsequent amplification, thereby reducing the time required for the complete enrichment process while maintaining the ability to detect unknown structural variations.
2Loss of time
If dual-target primer based amplification is used to enrich regions of interest, then the protocol time is reduced to less than 8 hours and complexity is lowered, but the capability to enrich sequences with unknown structural variations is lost
Solution Approach 1:
The patent employs universal primer extension complexes that can bind to both known and unknown target sequences. The first and second oligonucleotides are designed to hybridize to the target nucleic acid regardless of its sequence variations, enabling the system to enrich regions with unknown structural variations while maintaining a simplified protocol similar to dual-target primer based amplification.
Solution Approach 2:
The patent introduces primer extension complexes as intermediary structures that mediate between the oligonucleotide probes and the target nucleic acids. These complexes serve as capture intermediaries that can form with targets of unknown sequence, allowing the system to maintain versatility while using a time-efficient amplification approach.
3Adaptability or versatility
If single-target primer based amplification is used to enrich targets, then the ability to detect sequences with unknown structural variations is improved, but the protocol becomes slower and more complicated than dual primer based approaches
Solution Approach 1:
The patent merges the advantages of hybridization capture and primer based amplification by combining the capture capability of oligonucleotide hybridization with the speed and simplicity of primer extension. The first and second primer extension complexes are formed and captured in a unified process, reducing the complexity compared to single-target approaches while maintaining the ability to detect unknown structural variations.
4Quantity of substance
If variant allele enrichment is performed to lower sequencing costs, then the number of sequencing reads needed is reduced, but the enrichment method must be fast, simple, and cost-effective
Solution Approach 1:
The patent optimizes parameters such as oligonucleotide sequence design, hybridization conditions, and polymerase selection to achieve efficient variant allele enrichment. By carefully controlling these parameters, the method reduces the number of sequencing reads needed while maintaining a fast and simple protocol that is cost-effective for detecting variant alleles at low frequencies.
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 significantly reduces sequencing costs and time by enriching for variant alleles with high specificity and sensitivity, accommodating unknown structural variations, and improving sequencing efficiency.
Implementation Method 1
hybridizing a first oligonucleotide to a target nucleic acid in a library of nucleic acids
Implementation Method 2
extending the hybridized first oligonucleotide with a first polymerase, thereby producing a first primer extension complex
Implementation Method 3
extending the hybridized second oligonucleotide with a second polymerase, thereby producing a second primer extension complex comprising the target nucleic acid and the extended second oligonucleotide, thereby liberating the extended first oligonucleotide from the first primer extension complex
Implementation Method 4
amplifying the target nucleic acid with a third polymerase, a first amplification primer, and a second amplification primer
Data Source
AI summary
The present disclosure provides a method for enrichment of at least one target nucleic acid in a library of nucleic acids. This present disclosure is also directed to a faster and easier method of target capture using primer extension reactions that can improve ease of use, turnaround time, and variant allele specificity by designing target enrichment primers to specifically enrich library fragments based on the relative location of the variant base(s) in the primer, the utilization of polymerases with better priming specificity, designing the variant bases in the capture primer, designing the variant bases in the release primer, and/or designing variant specific primers to the both the plus and minus strands of the target library fragment.