Nucleic Acid Detection Assay with Carryover Prevention
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Solution Overview
Problem
Current molecular diagnostics for cancer and infectious diseases face challenges in distinguishing disease-specific nucleic acid markers from normal tissue markers, achieving high sensitivity and specificity, and are often costly and complex, especially when dealing with rare or low-abundance mutations and low sample volumes.
Innovation Solution
A method involving combined nuclease, ligation, and polymerase reactions with carryover prevention, using primary and secondary oligonucleotide primer sets and probes to detect target nucleotide sequences, copy numbers, transcript sequences, and methylated residues, and employing spatial multiplexing for relative quantification, similar to digital PCR, to identify and quantify nucleic acid changes in blood samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If combined nuclease, ligation, and polymerase reactions with carryover prevention are used, then measurement precision and reliability are improved, but device complexity and ease of manufacture worsen
Solution Approach 1:
The assay is divided into distinct modular steps: nuclease digestion step, ligation step, and polymerase amplification step. Each step uses specific enzymes and reagents that can be optimized independently, allowing complex detection functionality to be achieved through coordinated simple modules.
Solution Approach 2:
The method incorporates carryover prevention by adding dUTP to PCR products and using Uracil-DNA Glycosylase (UDG) to digest any carryover contaminant before the next amplification cycle. This preliminary cleanup action ensures that even if physical contamination occurs, the enzymatic barrier prevents false positives.
2Productivity
If spatial multiplexing is employed for relative quantification, then productivity is improved, but device complexity worsens
Solution Approach 1:
The assay achieves spatial multiplexing by incorporating multiple distinguishable elements into a single reaction well: different fluorescent labels, various enzyme specificities, and multiple target sequences. This allows parallel detection of multiple analytes in one well, effectively adding dimensional complexity to the reaction space rather than requiring separate physical wells.
3Reliability
If enzymes capable of digesting deoxyuracil containing nucleic acid molecules are used, then reliability is improved, but ease of operation worsens
Solution Approach 1:
The carryover prevention system is self-service in that the UDG enzyme automatically digests dUTP-containing contaminant DNA in the reaction mixture before amplification begins. The system monitors and corrects its own contamination issues without requiring external intervention or complex additional steps beyond the standard enzyme addition.
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 and cost-effective early detection of cancer and monitoring of disease recurrence with high sensitivity and specificity, reducing false positives and enabling the detection of low-abundance mutations, while maintaining modular and scalable design for clinical applications.
Implementation Method 1
contacting the sample with one or more enzymes capable of digesting deoxyuracil (dU) containing nucleic acid molecules present in the sample
Implementation Method 2
The contacted sample is blended with the one or more primary oligonucleotide primer sets, a deoxynucleotide mix including dUTP, and a DNA polymerase to form a polymerase chain reaction mixture
Implementation Method 3
The primary extension products are blended with a ligase and one or more oligonucleotide probe sets to form a ligation reaction mixture. The one or more oligonucleotide probes of the one or more oligonucleotide probe sets are ligated together to form ligated product sequences
Implementation Method 4
the first and second oligonucleotide probes of a probe set are configured to hybridize, in a base specific manner, on a complementary target nucleotide sequence of a primary extension product
Data Source
AI summary
The present invention relates to methods and devices for identifying and quantifying, including low abundance, nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicings, exon insertions, exon deletions, intron insertions, or other rearrangement at the genome level and/or methylated nucleotide bases.