Nucleic Acid Probe for Multiplexing and Selection Accuracy
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Solution Overview
Problem
Existing nucleic acid selection methods require careful selection of restriction enzymes to avoid cleavage within the target sequence, limiting multiplexing and leading to the amplification of unduly long nucleic acid fragments, increasing analysis costs.
Innovation Solution
A method using a single-stranded nucleic acid probe with four target-specific binding sites arranged in order, allowing for cleavage site creation without the need for prior fragmentation, enabling the selection and circularization of target sequences without specific probe binding sites, and facilitating precise selection and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If restriction enzymes are used to fragment nucleic acid prior to selection, then selection can be performed, but the choice of enzymes is limited and may cleave within the target sequence
Solution Approach 1:
The probe is designed with pre-positioned binding sites that flank the target sequence before selection. This preliminary arrangement of binding sites allows the probe to define cleavage boundaries without requiring prior enzymatic fragmentation, thereby avoiding enzyme selection limitations and preventing cleavage within the target sequence.
Solution Approach 2:
The nucleic acid probe acts as an intermediary element that mediates between the target sequence and the selection process. By providing defined binding sites that flank the target, the probe enables precise selection without directly requiring restriction enzymes to fragment the nucleic acid, thus resolving the contradiction between multiplexing capability and selection accuracy.
2Reliability
If restriction enzymes are carefully selected to avoid cleavage within target sequence, then selection accuracy is improved, but the degree of multiplexing is limited
Solution Approach 1:
The probe design provides a universal approach that can be applied to multiple target sequences simultaneously. By incorporating multiple binding sites that flank different target regions, a single probe system enables multiplexing while maintaining selection accuracy, as the probe-defined boundaries prevent enzyme cleavage within targets regardless of which targets are being selected.
Solution Approach 2:
The probe pre-establishes binding sites that flank target sequences before the selection process begins. This preliminary configuration allows multiple targets to be selected simultaneously with high accuracy, as the probe's binding sites define precise boundaries that prevent cleavage within any of the target sequences, thereby enabling both high multiplexing capability and selection accuracy.
3Ease of manufacture
If prior fragmentation is performed to create specific binding sites, then probe binding is enabled, but target fragments become unduly long increasing analysis costs
Solution Approach 1:
The probe is designed with binding sites that are positioned to flank the target sequence before the selection process. This preliminary arrangement eliminates the need for prior fragmentation to create binding sites, as the probe's pre-positioned sites naturally define the boundaries of the fragment to be selected, thereby avoiding the generation of unduly long fragments and reducing analysis costs.
Solution Approach 2:
The probe serves as an intermediary that provides the binding sites needed for selection without requiring prior fragmentation of the nucleic acid. By mediating the selection process through its pre-positioned binding sites, the probe enables precise fragment selection while avoiding the creation of excessively long fragments that would increase analysis costs.
4Adaptability or versatility
If multiple different primer pairs are used for multiplex assay, then multiple targets can be amplified simultaneously, but amplification artifacts occur
Solution Approach 1:
The probe design enables a universal amplification approach where a single primer pair can amplify multiple target sequences simultaneously. By incorporating multiple binding sites that flank different targets within the probe, the system achieves multiplexing without requiring multiple different primer pairs, thereby eliminating amplification artifacts associated with multiple primer sets while maintaining high multiplexing capability.
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 allows for precise selection and analysis of target nucleic acid sequences with reduced errors in sequencing, enabling efficient detection and amplification of rare mutations, and supports multiplexing without the need for specific restriction enzymes, thus reducing analysis costs and improving sequencing accuracy.
Implementation Method 1
a first target-binding site, complementary to a first outer flanking sequence flanking a first side of the ROI in the target molecule
Implementation Method 2
digesting any linear non-circularised nucleic acid molecules using exonuclease enzymes
Data Source
AI summary
The present invention relates to a method of selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising sequences capable of directing the cleavage of a target nucleic acid molecule to release a fragment comprising the ROI and sequences capable of templating the circularisation and ligation of the target fragment. The circularised molecule thus obtained contains the selected ROI and may be subjected to further analysis and/or amplification etc. Also provided are probes and kits for use in such methods.