Nucleic Acid Probe Circularization for Multiplexing
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Solution Overview
Problem
Existing nucleic acid selection methods require precise selection of restriction enzymes, limiting multiplexing and leading to amplification of unduly long nucleic acid fragments, increasing analysis costs.
Innovation Solution
A method using a nucleic acid probe with a 3′ sequence capable of hybridizing and extending to form a complement, and an internal sequence for circularization, allowing for the selection and amplification of target sequences without prior cleavage, thereby minimizing the need for precise restriction enzyme selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If restriction enzymes are used to cleave target nucleic acid prior to selection, then specific binding sites are created for probe selection, but the selection of enzymes is limited and can cleave within target sequences, leading to amplification of long fragments
Solution Approach 1:
The invention extracts the cleavage function from the selection process by using separate restriction enzyme digestion of the target nucleic acid before probe hybridization. This separates the fragment generation step from the selection step, allowing independent optimization of each process and enabling multiplexing without concern for enzyme sites within target sequences.
Solution Approach 2:
The method segments the nucleic acid selection process into distinct steps: (1) restriction enzyme cleavage of target DNA to generate fragments, (2) probe hybridization to selected fragments, and (3) circularization of probe-target hybrids. This segmentation allows each step to be optimized independently and facilitates multiplexing by allowing multiple probes to target different fragments simultaneously.
2Reliability
If restriction enzymes are carefully selected to avoid cleaving within target sequences, then target sequence integrity is maintained, but the degree of multiplexing is limited
Solution Approach 1:
The target nucleic acid is pre-cleaved by restriction enzymes before probe hybridization, generating a pool of fragments with known boundaries. This preliminary action ensures that subsequent probe selection only targets intact regions between restriction sites, maintaining sequence integrity while allowing multiple probes to operate simultaneously on different fragments.
3Productivity
If multiple different primer pairs are used for amplification of multiple target fragments, then simultaneous amplification is achieved, but amplification artefacts result
Solution Approach 1:
The invention makes the probe structure universal by incorporating a common primer binding site and circularization sequence in all probes, regardless of their target specificity. This allows all probe-target hybrids to be circularized and amplified using the same universal primers, enabling simultaneous amplification of multiple targets without the artefacts associated with multiple different primer pairs.
4Ease of manufacture
If the probe method does not require prior cleavage of sample nucleic acid, then restriction enzyme selection is minimized, but the method requires the nucleic acid to be in a form accessible to probe binding
Solution Approach 1:
Instead of requiring the target nucleic acid to have pre-existing binding sites for probes (as in traditional methods), this invention inverts the approach by having the probe bind to any single-stranded region and then using the probe sequence itself to define the selection boundaries through circularization. This eliminates the need for careful matching of restriction sites to probe locations.
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 enables more precise selection and amplification of target sequences without the need for specific binding sites, reducing analysis costs and improving multiplexing capabilities.
Implementation Method 1
a 3' sequence capable of hybridising to a target nucleic acid molecule
Implementation Method 2
acting as a primer for the production of a complement of the target ROI (i.e. by target templated extension of the primer)
Implementation Method 3
an internal sequence capable of templating the circularisation and ligation of the extended probe
Implementation Method 4
capable of templating the circularisation and ligation of the extended probe
Data Source
AI summary
The present invention relates to a method for selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising a 3′ sequence capable of hybridising to a target nucleic acid molecule and acting as a primer for the production of a complement of the target ROI (i.e. by target templated extension of the primer), and a sequence capable of templating the circularisation and ligation of the extended probe comprising the reverse complement of the target ROI and a portion of the probe. The circularised molecule thus obtained contains the reverse complement of the target ROI and may be subjected to further analysis and/or amplification etc. The probe may be provided as an oligonucleotide comprising a stem-loop structure or as a partially double-stranded construct and comprises a single-stranded 3′ end region containing the target-binding site. A second binding site provided in the probe serves as the ligation template for circularisation, and the stem-loop structure, if present, is cleaved to render the second binding site available for hybridisation to the target complement. Also provided are probes and kits for carrying out such a method.