Single-End Nucleic Acid Probe for Unknown Sequence Enrichment
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Solution Overview
Problem
Current methods for isolating or enriching target nucleic acids require knowledge of both end sequences, making it difficult to isolate genomic sequences with unknown end sequences, such as in analyzing chromosomal translocation breakpoints or splicing patterns, where only one region of sequence is known.
Innovation Solution
A method using a single target-specific probe that binds only once to a target nucleic acid fragment, allowing for single-sided ligation and enrichment without requiring sequence information from both ends, facilitated by oligonucleotides A and B that hybridize specifically to one end of the fragment, enabling covalent attachment and subsequent immobilization or amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If two target-specific probes are used to bind both ends of a target nucleic acid fragment, then the specificity and reliability of target isolation is improved, but the requirement for sequence knowledge increases and the method becomes inapplicable to targets with unknown end sequences
Solution Approach 1:
The invention extracts the target-specific binding function to only one end of the nucleic acid fragment, rather than requiring binding at both ends. This allows the method to work with targets where only one end sequence is known, while still maintaining sufficient specificity through the single end-specific probe combined with size selection
Solution Approach 2:
The invention makes the method universally applicable to both known and unknown target sequences by using a combination of one end-specific probe and size-based selection. This multi-functional approach allows the same basic protocol to work whether one or both end sequences are known, eliminating the limitation of requiring sequence information at both ends
2Reliability
If two target-specific hybridisation events are required for each target fragment, then the specificity of target detection is improved, but the complexity of the method increases and the ease of operation deteriorates
Solution Approach 1:
The invention removes one of the two required hybridisation events, simplifying the protocol to require only a single end-specific probe. The specificity previously provided by the second probe is replaced by size selection methods, making the procedure easier to execute while maintaining adequate specificity
Solution Approach 2:
The invention changes the selection parameter from sequence-specific hybridisation at both ends to a combination of single-end hybridisation and size-based selection. This parameter change simplifies the operational steps while maintaining the ability to specifically isolate target fragments
3Manufacturing precision
If stringent washing is applied to ensure high specificity, then the purity of isolated target nucleic acids is improved, but the risk of losing target material increases
Solution Approach 1:
The invention performs preliminary size selection before stringent washing to pre-enrich the target population. This preliminary action ensures that the majority of material present is target-related, allowing stringent washing to be applied with minimal loss of target material while still achieving high purity
Solution Approach 2:
The invention uses size selection as a partial pre-purification step that captures the bulk of target material before the stringent washing step. This partial action protects against excessive loss during washing by ensuring that non-target material has already been removed
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
Enables efficient detection and enrichment of target nucleic acids with unknown sequences at one end, providing high specificity through stringent washing and allowing analysis of complex samples, including degraded DNA, without the need for extensive sequence knowledge.
Implementation Method 1
oligonucleotide A is a single-stranded oligonucleotide comprising at one end a first target-specific part comprising at least 10 nucleotides complementary in sequence to at least part of said single-stranded portion of said target fragment
Implementation Method 2
ligating oligonucleotide B of said probe to the end of said target fragment which is hybridized to oligonucleotide A of the probe to produce a probe-target fragment hybrid
Implementation Method 3
the at least one element for enrichment in oligonucleotide B of the probe comprises an element for immobilization to a solid phase, and wherein enrichment in step (e) is by means of immobilization to a solid phase by means of the immobilization element
Implementation Method 4
providing high specificity through stringent washing
Data Source
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AI summary
The present invention provides a method for detecting or enriching for a target deoxyribonucleic acid (DNA) present in a nucleic acid sample, said method comprising: (a) fragmenting a nucleic acid sample to generate nucleic acid fragments including a target fragment containing said target DNA; (b) rendering said fragments, including said target fragment, at least partially single-stranded, wherein the single- stranded portion includes an end portion and wherein the length of said single- stranded portion is sufficient to allow hybridisation of at least part of the single- stranded portion of said target fragment to the probe of step (c); (c) contacting the at least partially single-stranded fragments of step (b) with oligonucleotides A and B of a single target-specific nucleic acid probe, wherein: (i) oligonucleotide A is a single- stranded oligonucleotide comprising at one end a first target-specific part comprising at least 10 nucleotides complementary in sequence to at least part of said single- stranded portion of said target fragment, and comprising at the other end a second non-target-specific part which comprises a nucleotide sequence complementary to at least a portion, including one end, of oligonucleotide B of the probe, and (ii) oligonucleotide B is a single-stranded oligonucleotide which may contain or carry at least one element for detection and/or enrichment of said target fragment, and of which at least a portion, including one end, is complementary in sequence to the second non-target-specific part of oligonucleotide A, such that said target fragment becomes annealed to said probe through hybridisation to the first target-specific part of oligonucleotide A resulting in only one target-specific probe-binding event per target fragment; (d) ligating oligonucleotide B of said probe to the part of the single- stranded portion of said target fragment which is hybridised to oligonucleotide A of said probe to produce a probe-target fragment hybrid; and (e) detecting or enriching for said probe-target fragment hybrid. Kits for use in the method of the invention are also provided.