In Situ RCA Synchronization for Uniform Signal Spot Detection
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Solution Overview
Problem
In situ assays face challenges with heterogeneous size and intensity distribution of signal spots due to unsynchronized rolling circle amplification (RCA) reactions, leading to overlapping large spots masking smaller ones and undetectable dim spots, which compromises sensitivity and resolution.
Innovation Solution
A method involving preformed complexes of polymerase and primers in an OFF buffer, followed by synchronization with an ON buffer to initiate RCA simultaneously across multiple locations, stabilizing the polymerase and inhibiting its activity until activation, ensuring uniform RCA product size and intensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If rolling circle amplification is performed without synchronization, then multiple analytes can be detected simultaneously, but heterogeneous size and intensity distribution of signal spots occurs leading to overlapping and masking
Solution Approach 1:
The patent applies preliminary action by pre-forming complexes between polymerase and primer in an OFF buffer before introducing the circular template. This pre-complex formation ensures that polymerase is already positioned and ready to initiate RCA simultaneously at multiple locations when the circular template is introduced, eliminating the heterogeneity in signal spot size and intensity that occurs with unsynchronized reactions.
Solution Approach 2:
The patent utilizes parameter changes by switching between two buffer conditions: an OFF buffer that inhibits polymerase activity to allow pre-complex formation, and an ON buffer that activates polymerase activity to initiate synchronized RCA. This buffer switching mechanism enables precise control over when and where amplification occurs, achieving uniform signal spot characteristics while maintaining simultaneous multi-analyte detection capability.
2Stability of the object's composition
If polymerase activity is inhibited during sample processing, then preformed complexes can be stabilized, but RCA reaction cannot proceed until activation
Solution Approach 1:
The patent applies dynamics by creating a system where polymerase activity is dynamically controlled through buffer conditions. The OFF buffer stabilizes the preformed polymerase-primer complex by inhibiting polymerase activity, while the ON buffer activates the complex to proceed with RCA. This dynamic switching between inhibited and active states enables both stable complex formation and subsequent productive amplification.
Solution Approach 2:
The buffer acts as an intermediary that mediates between the stable complex formation state and the active RCA reaction state. By introducing the buffer as a controllable parameter, the system can transition from a stable inhibited state (allowing complex formation) to an active state (allowing RCA progression), thus resolving the contradiction between stability and productivity.
3Quantity of substance
If unsynchronized RCA reactions occur at different locations, then analyte abundance variations are captured, but large signal spots mask adjacent smaller spots
Solution Approach 1:
The patent applies preliminary action by pre-forming polymerase-primer complexes at all locations before introducing the circular template. This ensures that when RCA is activated, all locations start amplification simultaneously rather than sequentially. The synchronized initiation prevents large signal spots from forming before smaller spots, thereby eliminating the masking problem while still capturing analyte abundance information through the circular template hybridization process.
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 results in more homogeneous RCA products with improved sensitivity and resolution by reducing the heterogeneity in size and intensity, allowing for better detection and analysis of analytes in biological samples.
Implementation Method 1
a rolling circle amplification product of the circular nucleic acid can be generated in the biological sample
Implementation Method 2
the chelating agent can chelate a di-cation such as Mg2+ from one or more prior reactions
Implementation Method 3
the circular nucleic acid or the polymerase is prebound to a polynucleotide comprising a sequence complementary to the hybridization region
Data Source
AI summary
The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.