Paired Probe Detection of Single and Double Stranded Nucleic Acids
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Solution Overview
Problem
Current nucleic acid detection methods are limited in distinguishing between single-stranded and double-stranded nucleic acids, particularly in complex environments like tissue samples, and lack sensitivity and multiplex capabilities for simultaneous detection of DNA and RNA.
Innovation Solution
The use of paired probes with different colored signals to distinguish between single-stranded and double-stranded nucleic acids, where sense and anti-sense probes are designed not to be complementary to avoid cross-hybridization, allowing for in-situ detection and characterization of DNA and RNA in their respective forms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional Southern blot analyses are used to detect nucleic acids, then detection capability is provided, but the method requires denaturation of nucleic acids and cannot distinguish between single-stranded and double-stranded forms
Solution Approach 1:
The detection system is segmented into multiple probe types (first probes complementary to sense strands, second probes complementary to anti-sense strands) that can independently bind to different strands of nucleic acids. This segmentation allows the system to detect and distinguish between single-stranded and double-stranded forms by observing which probe combinations produce signals, thereby resolving the contradiction between detection capability and strand structure information preservation.
2Measurement precision
If Northern blot, dot blot, and nuclease protection assays are used to detect RNA, then detection is possible, but the methods are time-consuming, have limited sensitivity, and produce more qualitative than quantitative data
Solution Approach 1:
The invention merges multiple detection capabilities into a single hybridization-based system that can simultaneously detect, quantify, and characterize nucleic acids. By combining the probe hybridization mechanism with signal detection systems, the method achieves both speed and quantitative precision, resolving the contradiction between detection capability and experimental time consumption.
3Measurement precision
If PCR and RT-PCR based methods are used for detection and quantitation, then sensitivity and quantification are improved, but multiplex capabilities are limited
Solution Approach 1:
The probe system is designed with universal applicability through multiple probe types that can detect various nucleic acid forms (single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA) using the same hybridization platform. This multi-functionality enables simultaneous detection of different nucleic acid types and forms, resolving the contradiction between sensitivity/quantification and multiplex capabilities.
4Measurement precision
If in situ hybridization methods are used to detect nucleic acids in complex environments, then localization is possible, but the methods cannot simultaneously detect DNA and RNA or confirm double stranded status
Solution Approach 1:
The detection system uses segmented probe strategies where first probes target sense strands and second probes target anti-sense strands. By observing which probe combinations produce signals at specific locations, the system can simultaneously determine nucleic acid type (DNA vs. RNA) and structure (single-stranded vs. double-stranded) while maintaining localization capability, thus resolving the contradiction between localization and comprehensive information detection.
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 enables accurate localization and quantitation of nucleic acids, improving sensitivity and specificity, and allows for simultaneous detection of DNA and RNA in the same sample, overcoming the limitations of existing methods.
Implementation Method 1
paired probes provide signals that distinguish single stranded target nucleic acids from double stranded nucleic acids
Implementation Method 2
the first and second reporters provide signals of different colors, whereby the paired colors are readily distinguished by the human eye
Data Source
AI summary
This invention provides compositions, methods, and systems for characterizing, resolving, and quantitating single stranded and double stranded DNA and RNA in-situ. Paired sense and anti-sense probes can signal the presence of double stranded nucleic acids. DNA and RNA can be distinguished in cell and tissue samples by hybridizing with probe sets adapted to highlight differences in these targets in-situ.