Paired Primer Panels for RNA Detection in Degraded Samples
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Solution Overview
Problem
Current methods for detecting and sequencing RNA or DNA in samples with low concentrations or poor quality, such as degraded or historical samples, face challenges with low titer, cryptic sequences, and limited sample quantity, leading to incomplete or unsuccessful phylogenetic data generation.
Innovation Solution
The use of paired large panels of primers to amplify many short overlapping fragments, followed by a preliminary multiplex amplification step, enables complete gene or genomic coverage, allowing for the detection and sequencing of RNA or DNA in challenging samples, including formalin-fixed paraffin-embedded and blood samples, even when conventional methods fail.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RT-PCR assays are used to amplify short conserved sequences, then detection sensitivity is improved, but phylogenetic data utility is lost
Solution Approach 1:
The patent segments the genome into multiple overlapping amplicons of moderate length (200-500 bp) that can be individually amplified and sequenced. This segmentation allows each fragment to be sufficiently long for phylogenetic analysis while being short enough to amplify from degraded samples, resolving the contradiction between detection sensitivity and phylogenetic utility.
Solution Approach 2:
The patent transitions from a single-dimension approach (amplifying one long fragment) to a multi-dimensional strategy by amplifying multiple overlapping fragments across the genome. This dimensional expansion enables both sensitive detection through multiple targets and comprehensive phylogenetic analysis through genome-wide coverage.
2Length of stationary object
If long PCR fragments (>1000 bp) are amplified to generate phylogenetic data, then sequence length is improved, but sample quality requirements increase
Solution Approach 1:
Instead of attempting to amplify one long fragment (>1000 bp) that requires high sample quality, the patent segments the genome into multiple shorter overlapping amplicons (200-500 bp). Each segment can be reliably amplified from degraded samples while the collective set provides sufficient sequence length for phylogenetic analysis.
Solution Approach 2:
The patent uses an excessive number of primer pairs (50-200) to ensure that enough amplicons are successfully amplified from degraded samples. This partial amplification approach, where not all fragments need to be recovered, ensures phylogenetic utility even when sample quality is poor.
3Loss of information
If NGS library technology is used to develop long sequences from fractured specimens, then sequence recovery is improved, but minimum RNA titer requirements increase
Solution Approach 1:
The patent segments the genome into multiple short amplicons that can each be amplified from low-titer samples. By distributing the amplification demand across many short targets rather than requiring amplification of long fragments, the method achieves effective sequence recovery from samples with RNA titers below the 100-200 ng minimum required by conventional NGS.
Solution Approach 2:
The patent changes the key parameters of the amplification strategy: using many short amplicons (200-500 bp) instead of few long fragments, and employing 50-200 primer pairs instead of standard NGS library preparation. These parameter changes enable successful amplification and sequencing from samples with RNA titers as low as 1-10 ng, far below conventional NGS requirements.
4Ease of operation
If a single primer pair is used for RT-PCR, then assay simplicity is maintained, but detection of cryptic sequences fails
Solution Approach 1:
The patent creates a universal amplification system using 50-200 primer pairs that can detect and amplify diverse viral sequences including cryptic variants. This multi-functional primer set maintains operational simplicity by using a standardized protocol while dramatically increasing adaptability to detect unknown or divergent sequences that would fail with a single primer pair.
Solution Approach 2:
The patent uses an excessive number of primer pairs (50-200) to ensure coverage of cryptic sequences. This partial redundancy approach, where many primers are used beyond what a single assay would require, ensures that at least some primers will bind to divergent sequences, maintaining both simplicity and versatility.
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 successfully generates near full-length sequences from previously unusable samples, providing high sensitivity and universal applicability for RNA and DNA detection, including rare variants and historical pathogens like HIV, with scalability and automation potential.
Implementation Method 1
The method comprises subjecting the sample to reverse transcription (RT) using reverse transcriptase and one primer from each of one or more pairs of primers
Implementation Method 2
The method may further comprise subjecting the sample from the previous step to polymerase chain reaction (PCR) amplification using Taq polymerase and the other primer of each of the one or more pairs of primers
Data Source
AI summary
Disclosed are methods and systems for detecting RNA and sequencing RNA in a wide range of samples such as samples with low concentrations of nucleic acid, samples with degraded nucleic acid, samples that would not otherwise be amenable to conventional sequencing or RNA detection methods, poor quality samples, high quality samples in which rare mutations are sought, formalin-fixed paraffin-embedded samples, blood samples, etc. The methods of the present invention may use paired, large panels of primers to amplify many short fragments that overlap between but not within each panel. Each panel's amplicon set may fill the gaps between those of the opposing panel, thereby providing complete gene or genomic coverage. A preliminary, multiplex amplification step amplifies target nucleic acid for all downstream reactions such as Sanger sequencing, cloning, and Next Generation Sequencing (NGS).