Orthogonal Six-Base Universal Sequences for bDNA Assays
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Solution Overview
Problem
The existing branched DNA (bDNA) assays face challenges with background noise due to non-specific hybridization, particularly in multiplex formats, which reduces sensitivity and makes it difficult to accurately detect and genotype viruses, retroviruses, SNPs, and cytokines.
Innovation Solution
Development of highly orthogonal six-base universal sequences comprising four natural bases and two non-natural bases, such as isoguanosine and isocytosine, which are designed to minimize cross-hybridization, with a melting temperature of approximately 80-85°C, allowing for specific binding and reduced non-specific interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional four-base universal sequences are used in bDNA assays, then the assay can detect targets, but background noise increases due to non-specific hybridization
Solution Approach 1:
The patent changes the chemical parameter of the base pairing system by introducing two non-natural bases (iso-G and iso-C) that form Watson-Crick base pairs with each other but do not hybridize with natural bases. This parameter change creates a chemically distinct hybridization system that eliminates cross-reactivity between universal sequences and target sequences, thereby reducing background noise while maintaining detection capability
Solution Approach 2:
The patent creates a composite hybridization system that combines four natural bases (A, T, G, C) and two non-natural bases (iso-G, iso-C) in the same oligonucleotide sequence. This composite material approach allows the universal sequence to contain both natural bases for binding to the solid support and non-natural bases for specific binding to target sequences, while preventing non-specific hybridization through the orthogonal chemistry of iso-G/iso-C pairs
2Reliability
If six-base universal sequences with non-natural bases are used, then cross-reactivity is minimized, but sequence design and synthesis complexity increases
Solution Approach 1:
The patent segments the oligonucleotide sequence into distinct functional regions: a 5' region containing natural bases for solid support binding, a middle region with non-natural iso-G/iso-C bases for specific target binding, and a 3' region with natural bases for probe binding. This segmentation allows each region to be optimized independently for its specific function while maintaining overall sequence orthogonality
Solution Approach 2:
The patent applies local quality by assigning different base compositions to different regions of the oligonucleotide. The 5' end uses natural bases for general binding, the central portion uses non-natural iso-G/iso-C bases for specific orthogonal interactions, and the 3' end uses natural bases for probe hybridization. This local differentiation optimizes each region's function while maintaining overall sequence performance
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
The use of these universal sequences significantly improves the accuracy and specificity of bDNA assays by minimizing cross-reactivity, enabling the detection of low viral loads and precise genotyping of SNPs, and enhancing the sensitivity of cytokine mRNA quantitation.
Implementation Method 1
The bDNA assay is used to quantify RNA and DNA targets from a variety of sources. The sensitivity and specificity of the assay are derived in part through the judicious choice of oligonucleotide probes that constitute the probe set.
Implementation Method 2
Iso-G and iso-C form standard Watson and Crick interactions with each other; however, because the hydrogen bonding pattern between the iso-G and iso-C is different from the hydrogen bonding pattern between the natural bases, there is no interaction between iso-G and iso-C and the natural bases.
Implementation Method 3
Iso-G and iso-C form standard Watson and Crick interactions with each other; however, because the hydrogen bonding pattern between the iso-G and iso-C is different from the hydrogen bonding pattern between the natural bases, there is no interaction between iso-G and iso-C and the natural bases.
Implementation Method 4
The DNA or RNA targets are labeled using a large number (typically>30) target-specific oligonucleotides called label extender (LE) probes, which mediate the hybridization of bDNA amplifier molecules to the CEs.
Data Source
AI summary
The invention provides a set of highly orthogonal six-code universal sequences for use in bDNA singleplex and multiplex nucleic acid hybridization assays. The six-code orthogonal sequences do not cross-hybridize and thus, minimize or eliminate the 3-mer cross-hybridization inherent in the second and third generation bDNA assays. The highly orthogonal universal sequences may be used in singleplex or multiplex bDNA assays quantitatively and qualitatively to determine mRNA levels in a sample; to screen for and genotype targets, such as viruses, that are present in low volumes in a sample; to screen for and genotype SNPs; and to measure changes in the amount of a gene in a sample such as when gene amplifications or deletions occur. The highly orthogonal universal sequences may also be used as universal capture probes to selectively bind assay components in a way that facilitates their further analysis.