Nucleic Acid Quantification via Enzymatic Normalization
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Solution Overview
Problem
Current methods for quantifying nucleic acid modifications, such as methylation states, in mixed samples are inaccurate due to inefficiencies in enzymatic reactions, leading to incomplete and unreliable results, especially when dealing with small sample quantities.
Innovation Solution
The use of digital analysis and multiplexed assays that involve enzymatic reactions with control nucleic acid molecules to normalize the quantification of target regions, allowing for the differentiation between methylation states and improving the accuracy of nucleic acid quantification by measuring the efficiency of the enzymatic reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional enzymatic reactions are used to quantify nucleic acid modifications, then the quantification process is simple, but the accuracy and reliability of results deteriorate due to enzymatic reaction inefficiencies
Solution Approach 1:
The patent introduces control nucleic acid molecules as intermediary elements that mediate between the enzymatic reaction process and the final quantification measurement. These control molecules serve as internal references that allow normalization of enzymatic reaction efficiency, thereby improving measurement precision and result reliability without complicating the overall quantification approach
2Measurement precision
If control nucleic acid molecules are added to normalize quantification, then measurement precision improves, but device complexity increases due to multiplexed assays
Solution Approach 1:
The patent merges the quantification of target nucleic acid molecules with control nucleic acid molecules into a single multiplexed enzymatic reaction system. By combining these measurements in one assay rather than performing separate reactions, the patent achieves improved measurement precision while minimizing the increase in procedural complexity
3Quantity of substance
If small sample quantities are used, then the non-invasive nature of the test is maintained, but measurement precision deteriorates due to insufficient starting material
Solution Approach 1:
The control nucleic acid molecules act as intermediary reference standards that enable accurate quantification even when starting with small sample quantities. By normalizing the enzymatic reaction efficiency using these control molecules, the system can reliably measure low concentrations of fetal DNA in maternal blood without requiring large sample volumes
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 enhances the sensitivity and precision of nucleic acid quantification, enabling the accurate identification of disease states or conditions by normalizing the measured molecules based on enzymatic reaction efficiency, even in samples with low concentrations of modified nucleic acids.
Implementation Method 1
species of methylation-specific restriction enzymes (also referred to as MSRE) provide a mechanism for enrichment of a population of nucleic acid molecules by utilizing the characteristics of one or more MSRE species that includes an inability to cleave nucleic acid molecules at the enzyme recognition site when in a particular methylation state
Data Source
AI summary
In certain aspects, methods of the invention involve performing modification state specific enzymatic reaction of nucleic acid in a sample, determining a value associated with efficiency of the modification state specific enzymatic reaction based on a control, determining an amount of target nucleic acid in the sample, and normalizing the amount of target nucleic acid based on the efficiency value. Based on the normalized amount of target nucleic acid, the method further includes determining whether the normalized amount of target nucleic acid is indicative of a condition.