Clamp Oligonucleotide Circularization for Selective Nucleic Acid Capture
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Solution Overview
Problem
Current nucleic acid amplification methods lack efficient and selective capture mechanisms for target nucleic acids, which hinders purification, amplification, and sequencing processes.
Innovation Solution
The use of clamp oligonucleotides with user-defined tether regions containing primer sites, anchor sites, and barcodes allows for the immobilization and amplification of target nucleic acids by forming circularized complexes that can be captured and manipulated using polymerase extension and ligase joining, enabling further amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional nucleic acid amplification methods are used, then amplification can be performed, but efficient and selective capture mechanisms are lacking which hinders purification and sequencing processes
Solution Approach 1:
The clamp oligonucleotide is divided into distinct functional segments: target-binding regions (for specific target capture), tether regions (containing capture elements and primer-binding sites), and structural elements. This segmentation allows each region to perform its specific function optimally, enabling both selective capture and efficient downstream processing
Solution Approach 2:
The clamp oligonucleotide structure integrates multiple functions into a single molecule: target binding, capture element presentation, and primer-binding site provision. This multi-functionality eliminates the need for separate capture and amplification reagents, thereby improving both selective capture efficiency and overall productivity
2Reliability
If clamp oligonucleotides with user-defined tether regions are used, then selective capture and amplification are enabled, but the method complexity increases compared to conventional approaches
Solution Approach 1:
The invention merges target binding, capture, and amplification initiation functions into a single clamp oligonucleotide structure. By combining these functions that would traditionally require separate reagents and steps, the method achieves high specificity while reducing overall procedural complexity
3Productivity
If circularized nucleic acid complexes are formed and captured, then amplification and sequencing can proceed efficiently, but additional steps are required compared to direct amplification methods
Solution Approach 1:
The clamp oligonucleotide performs preliminary actions by pre-binding to the target sequence and presenting both capture elements and primer-binding sites in a predetermined configuration. This preliminary organization of functional elements streamlines subsequent amplification and sequencing steps, improving productivity despite the additional capture step
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 enables selective capture and amplification of target nucleic acids, facilitating subsequent manipulations such as PCR, detection, and next-generation sequencing, improving the efficiency and specificity of nucleic acid handling.
Implementation Method 1
The first and second target binding regions of the clamp oligonucleotide are annealed to the target nucleic acid
Implementation Method 2
The clamp oligonucleotide is extended by polymerase from its 3'-terminus to its 5'-terminus to produce a first duplex nucleic acid
Implementation Method 3
The ends of the first nucleic acid are joined by ligase to produce a circularized nucleic acid
Implementation Method 4
The circularized nucleic acid is removed from the target nucleic acid and then captured by a capture means provided on a solid support
Data Source
AI summary
The present invention provides methods of amplifying a target nucleic acid utilizing a clamp oligonucleotide comprising a first target-binding region on the 3′-terminus and a second target-binding region on the 5′-terminus and tether region in between. The tether region may comprise a variety of user-defined sequences or elements that allow for further manipulation of the target nucleic acid. Such as, for example, capture followed by amplification, identification and/or sequencing. The target-binding regions bind to the target nucleic acid, the 3′-terminus functions as a primer to initiate extension across the target nucleic acid sequence and ligation of the gap results in formation of a circularized nucleic acid. This circular template can be used in a variety of processes, including amplification and sequencing.