Expander Oligonucleotides for Degraded Nucleic Acid Detection
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Solution Overview
Problem
Formalin-fixed, paraffin-embedded (FFPE) samples are challenging for PCR analytical applications due to chemical modification and substantial degradation of nucleic acids, making it difficult to detect and quantify target nucleic acids, especially RNA, which are often degraded into short fragments.
Innovation Solution
The use of expander oligonucleotides, such as splint and prosthetic molecules, that convert target nucleic acid fragments into discretely sized DNA fragments, allowing for the simultaneous detection and quantification of DNA and RNA molecules, including partially degraded ones, by hybridizing with specific reverse-transcription primers and extending with template-dependent polymerases to produce surrogate markers of distinct lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR methods are used on FFPE samples, then the analysis can be performed, but the nucleic acids are substantially degraded and chemically modified making reliable detection difficult
Solution Approach 1:
The method segments the nucleic acid detection process into multiple stages: (1) reverse transcription of RNA to cDNA using random hexamers, (2) expansion of the cDNA using expander oligonucleotides to create discrete-length products, and (3) detection of the expanded products. This segmentation allows each stage to address specific degradation issues separately, with the expansion step creating standardized-length products from fragmented templates.
Solution Approach 2:
The method changes the length parameter of the nucleic acid products through the expansion step, transforming variable-length degraded fragments into discrete, standardized-length products. This parameter change enables reliable detection by creating uniform product sizes that can be consistently measured, overcoming the variability introduced by degradation.
2Adaptability or versatility
If RNA is extracted from FFPE specimens, then gene expression analysis can be performed, but the RNA is degraded into short fragments with loss of poly-A tracts
Solution Approach 1:
The method uses expander oligonucleotides as intermediary molecules that bind to the degraded RNA fragments and serve as templates for creating expanded products. These intermediaries bridge the gap between the degraded templates and the detectable products, allowing RNA detection despite fragment length reduction and poly-A tract loss.
Solution Approach 2:
The method performs reverse transcription of RNA to cDNA as a preliminary action before the expansion step. This preliminary conversion creates a stable cDNA intermediate that can then be expanded to discrete lengths, preserving information from the original RNA even though the RNA itself is degraded.
3Adaptability or versatility
If multiple nucleic acid types are detected simultaneously, then comprehensive analysis is achieved, but the complexity of the reaction increases
Solution Approach 1:
The method uses universal random hexamer primers for reverse transcription that can bind to various RNA sequences, and universal expander oligonucleotides that can expand different cDNA templates. This universality allows simultaneous detection of multiple nucleic acid types in a single reaction without requiring separate specialized reagents for each target.
Solution Approach 2:
The method merges the detection of multiple nucleic acid types into a single integrated reaction by combining reverse transcription, expansion, and detection steps into one workflow. This consolidation reduces the number of separate operations needed while maintaining the ability to detect multiple targets simultaneously.
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
Enables the reliable detection and quantification of multiple target nucleic acid sequences, including degraded RNA, in a single reaction, using size-based separation methods like capillary electrophoresis, overcoming the limitations of FFPE sample degradation.
Implementation Method 1
hybridizing with specific reverse-transcription primers and extending with template-dependent polymerases
Implementation Method 2
extending with template-dependent polymerases to produce surrogate markers of distinct lengths
Implementation Method 3
using size-based separation methods like capillary electrophoresis
Data Source
AI summary
Described herein are approaches for the detection, identification, and/or quantification of target nucleic acids, including, but not limited to partially, substantially randomly, degraded target nucleic acids, in a biological sample, such as a formalin-fixed, paraffin-embedded (FFPE) sample. These approaches provide a means of detecting, identifying, and/or quantifying target nucleic acid molecules, including DNA and RNA molecules, further including RNAs of different classes, from the same sample, and in the same reaction, by using “expander oligonucleotides,” as the term is defined herein, to convert fragments of target nucleic acids into discretely sized DNA fragments, each with a chosen length characteristic for the target nucleic acid from which it is derived.