Heteroduplex Binding Protein Isolation for Rare Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid separation techniques struggle to isolate rare nucleic acids, such as those with point mutations, from background nucleic acids, especially when the target nucleic acid is present in low abundance, limiting their use in early disease detection and analysis.
Innovation Solution
The method employs heteroduplex binding proteins, like MutS, to selectively bind to heteroduplex nucleic acids, allowing for their separation from homoduplex nucleic acids using time-varying driving and mobility fields, enabling the isolation of target nucleic acids even when they are present in very low concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard nucleic acid separation techniques are used, then the process is simple and widely applicable, but the ability to isolate rare nucleic acids from background nucleic acids is insufficient
Solution Approach 1:
The patent introduces heteroduplex binding proteins as intermediary molecules that specifically recognize and bind to heteroduplex nucleic acids containing mutations. These proteins act as mediators between the target rare nucleic acids and the separation system, enabling highly selective isolation of mutant sequences from abundant wild-type background through affinity-based enrichment before separation
Solution Approach 2:
The method performs preliminary enrichment of rare nucleic acids by incubating the sample with heteroduplex binding proteins before separation. This preliminary action selectively binds target heteroduplexes, concentrating them from the background and preparing them for subsequent high-resolution separation, thereby improving detection sensitivity without requiring complex separation conditions
2Measurement precision
If highly-specific ligands such as hybridization probes or antibodies are used to separate rare nucleic acid, then the separation precision is improved, but the method is not generally applicable to unknown target nucleic acid
Solution Approach 1:
The patent employs heteroduplex binding proteins that recognize a universal structural feature of all heteroduplex nucleic acids - the mismatched base pairing configuration. This universal recognition mechanism allows a single protein type to bind various different mutation sequences regardless of their specific identity, providing both high separation precision and broad adaptability to unknown mutations
Solution Approach 2:
The method exploits the structural parameter change that occurs when wild-type and mutant nucleic acids form heteroduplexes. The mismatched base pairing creates a distinct structural conformation that heteroduplex binding proteins specifically recognize. This parameter-based recognition (structural conformation rather than sequence identity) enables universal detection of any mutation type
3Measurement precision
If individual probes are required for each mutation, then the specificity for known mutations is high, but the ability to search for many known mutations at once is limited
Solution Approach 1:
The patent merges the function of multiple individual mutation-specific probes into a single heteroduplex binding protein system. Instead of requiring separate probes for each mutation, the method uses one type of protein that simultaneously recognizes all heteroduplexes in the sample, enabling parallel detection of multiple mutations in a single experiment and dramatically increasing screening throughput
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 allows for the effective isolation and recovery of target nucleic acids from a background of similar nucleic acids, even when they comprise only a small fraction, facilitating early disease detection and analysis without the need for prior amplification, which reduces the risk of introducing errors.
Implementation Method 1
The heteroduplex binding protein preferentially binds to heteroduplexed nucleic acids, creating a heteroduplex binding protein-nucleic acid complex that has a different mobility in a separation medium as compared to homoduplex nucleic acids
Implementation Method 2
separation of the heteroduplex and homoduplex nucleic acids using time-varying separation fields
Data Source
AI summary
The invention includes methods and apparatus for separating mutations, especially rare and unknown mutations, using heteroduplex binding proteins. Nucleic acids may optionally be nicked at or near the mutation in order to promote heteroduplex binding protein recognition and binding. In particular, using the disclosed methods, it is possible to separate heteroduplexed nucleic acid strand pair from homoduplexed nucleic acid strand pairs having similar sequences and being at a much higher concentration. Once the heteroduplexed nucleic acids are isolated and recovered, it is straightforward to analyze the sequences of the heteroduplexed nucleic acids, e.g., using sequencing or hybrid assays.