Single Probe Primer Extension for Target Enrichment
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Solution Overview
Problem
Current target enrichment methods for nucleic acid sequencing are either too complex and time-consuming or unable to accommodate unknown structural variations, lacking a fast and simple solution that can efficiently enrich sequences with such variations.
Innovation Solution
A method involving primer extension with a target-binding site and unique molecular identification tags, followed by ligation of adaptors with universal priming sites, and subsequent amplification using universal primers to create an amplified target sequence, which includes a purification step and the use of barcodes for molecular identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If hybridization capture is used to enrich target sequences, then the ability to capture exome-sized regions and regions with unknown structural variations is improved, but the protocol time and complexity increase significantly (taking well over 8 hours with hybridization alone taking up to three days)
Solution Approach 1:
The patent extracts the essential function of target enrichment by removing the time-consuming hybridization step and replacing it with a direct primer extension approach. The method takes out the unnecessary complexity of random fragmentation and hybridization while retaining the core capability to enrich target sequences, including those with unknown structural variations.
Solution Approach 2:
The patent performs preliminary actions by pre-designing primers with unique molecular identification tags and universal ligation sites before the enrichment process. This preliminary preparation allows the method to skip the time-consuming hybridization step while maintaining the ability to capture diverse target regions.
2Loss of time
If dual primer based amplification is used to enrich target sequences, then the protocol time is reduced to less than 8 hours and complexity is simplified, but the ability to enrich sequences with unknown structural variations is lost
Solution Approach 1:
The patent segments the primer into distinct functional regions: a target-binding site for specific sequence recognition, a unique molecular identification tag for tracking individual molecules, and a universal ligation site for adaptor attachment. This segmentation allows single primer amplification to achieve the versatility of dual primer methods while maintaining fast protocol times.
Solution Approach 2:
The patent implements universality by designing primers with universal ligation sites that can bind to various adaptors, and by using a single primer that can amplify multiple target regions with unknown structural variations. This multi-functional design enables the method to be both fast and versatile.
3Adaptability or versatility
If single-target primer based amplification with random fragmentation is used, then the ability to detect sequences with unknown structural variations is improved, but the protocol remains slower and more complicated than dual primer approaches
Solution Approach 1:
The patent extracts and removes the unnecessary random fragmentation step from single-target primer amplification. By directly amplifying targets from intact or minimally processed DNA using a single primer with target-binding capability, the method maintains versatility for detecting structural variations while dramatically simplifying the protocol.
Solution Approach 2:
The patent performs preliminary design of primers with built-in unique molecular identification tags and universal ligation sites, eliminating the need for post-amplification processing steps. This preliminary preparation simplifies the overall protocol while maintaining the ability to detect sequences with unknown structural variations.
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 PCR bias, allows for the detection of sequences with unknown structural variations, and provides a faster and simpler method for target enrichment, enabling efficient sequencing while minimizing complexity and time.
Implementation Method 1
conducting a polymerase extension reaction and a termination to create a single-stranded primer extension product
Implementation Method 2
ligating adaptors to each end of the single-stranded primer extension product to create a ligation product
Data Source
AI summary
The invention comprises methods and compositions for enriching for a target nucleic acid with a single primer extension and low-bias limited amplification.