Molecular Inversion Probe Assay Strand Displacement Error Reduction
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Solution Overview
Problem
Molecular inversion probes (MIPs) face inefficiencies due to polymerase strand displacement errors during the gap-fill step, leading to reduced assay signal strength and challenges in separating unreacted probes from circularized ones, which affects sensitivity and accuracy in nucleic acid detection.
Innovation Solution
The use of specifically designed MIPs with longer homology regions and cleavage substrates, along with enzymes like ribonucleases and mismatch endonucleases, to prevent strand displacement and facilitate efficient ligation without the need for a gap-fill reaction, and the incorporation of affinity pairs for selective removal of unhybridized probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a polymerase gap-fill reaction is used to close the nick in the MIP, then the MIP can be circularized for detection, but the polymerase may displace the 5′ end of the probe creating an undesirable extension that prevents ligation and reduces assay signal
Solution Approach 1:
The patent extracts and removes the problematic 5′ end extension created by polymerase strand displacement. A flap endonuclease is used to specifically cleave and remove the displaced 5′ end flap, eliminating the harmful byproduct that prevents ligation and reduces assay signal while preserving the desired gap-fill incorporation.
Solution Approach 2:
The patent introduces a flap endonuclease as an intermediary enzyme that mediates between the polymerase gap-fill reaction and the ligation step. This intermediary selectively removes the harmful 5′ end extension without affecting the correctly incorporated nucleotides, enabling subsequent successful ligation and circularization of the MIP.
2Ease of manufacture
If standard MIP protocols are used, then the assay can be performed with conventional reagents, but unreacted probes cannot be efficiently separated from circularized probes affecting sensitivity and accuracy
Solution Approach 1:
The patent applies local quality by incorporating a biotin tag at a specific location on the MIP probe. This localized modification creates a functional difference between unreacted probes (which retain the biotin tag) and circularized probes (which have the tag removed during circularization), enabling selective separation through streptavidin binding while maintaining overall assay simplicity.
3Productivity
If the MIP 5′ end is displaced by polymerase extension, then the gap-fill reaction proceeds, but the hybridized MIP cannot act as a ligase substrate and is not circularized for detection
Solution Approach 1:
The patent converts the harmful strand displacement extension into a beneficial intermediate structure. The displaced 5′ end creates a flap structure that is specifically recognized and cleaved by flap endonuclease, which then enables the ligation step to proceed efficiently by restoring the proper 5′ end for ligase substrate formation.
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 enhances the sensitivity, resolution, and accuracy of MIP analyses by reducing strand displacement errors and improving the separation of hybridized and unhybridized probes, leading to more reliable detection of target nucleic acids.
Implementation Method 1
Molecular inversion probes (MIPs) are, e.g., nucleic acid hybridization probes that hybridize to a target nucleic acid in a loop with the 5′ and 3′ ends abutting or separated in the target with a small gap
Implementation Method 2
If provided with the appropriate dNTP, the polymerase can fill the gap between the MIP 5′ and 3′ ends. For example, if the target nucleic acid has an adenine 'A' in the gap, the polymerase can fill the gap if provided with a complementary dTTP
Implementation Method 3
With the gap filled, a ligase can close the remaining nick and circularize the MIP
Implementation Method 4
Because circularized single strand DNA is not a substrate for many nucleases, all other nucleic acids, including MIPs that did not hybridize and circularize, can be digested with a nuclease cocktail
Implementation Method 5
A method includes providing an MIP with a 5′ end specifically removable when the MIP is hybridized to a target nucleic acid; hybridizing the MIP to the target; and specifically cleaving the 5′ end from the hybridized MIP
Data Source
AI summary
The invention provides methods and compositions to enhance the efficiency and sensitivity of molecular inversion probe (MIP) reactions. Probes include elements that allow MIP ends to abut for ligation while avoiding the possibility of polymerase strand displacement errors. Elements facilitate multiplexed detections, including MIP reaction product detections employing next generation sequencing (NGS) techniques.