DNA Sample Categorization via Signal Ratios
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Solution Overview
Problem
Current methods for categorizing and profiling DNA samples are often complex, require external controls, and struggle with accuracy and cost-effectiveness, particularly in distinguishing between different types of DNA and identifying their sources without relying on actual methylation levels.
Innovation Solution
A method involving digestion of DNA with methylation-sensitive and methylation-dependent restriction endonucleases, followed by PCR amplification of specific genomic loci, calculating signal ratios, and comparing these ratios to reference values to categorize DNA samples without the need for external controls, enabling simultaneous categorization and profiling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for categorizing and profiling DNA samples are used, then DNA samples can be categorized and profiled, but the methods are complex and require external controls
Solution Approach 1:
The invention extracts and eliminates the requirement for external controls and complex normalization procedures from the DNA categorization method. By using signal ratios between different loci within the same sample, the method removes the need for external reference DNA species and controls, thereby simplifying the overall procedure while maintaining categorization accuracy
Solution Approach 2:
The method enables the DNA sample to serve its own categorization needs through internal reference loci. The sample contains both target loci for categorization and reference loci that are constitutively unmethylated, allowing the sample to self-normalize and provide accurate categorization without requiring external controls or additional DNA species
2Ease of manufacture
If current methods for categorizing and profiling DNA samples are used, then DNA samples can be categorized, but external controls are required which increases cost and simplifies the assay
Solution Approach 1:
The PCR amplification step serves multiple functions simultaneously: it amplifies target loci for categorization, amplifies reference loci for internal normalization, and enables DNA profiling when combined with appropriate primers. This multi-functionality eliminates the need for separate control reactions and simplifies the overall assay procedure
Solution Approach 2:
The invention merges the categorization assay with DNA profiling into a single reaction. By using primer sets that amplify both categorization-specific loci and standard forensic STR loci in the same PCR reaction, the method reduces the number of separate assays needed and eliminates the requirement for external controls in separate reactions
3Measurement precision
If signal ratios are calculated and compared to reference values, then DNA categorization accuracy increases, but the method requires comparing multiple loci signals
Solution Approach 1:
The method assigns specific functional qualities to different loci: target loci that are differentially methylated in different DNA categories and reference loci that are constitutively unmethylated. This local differentiation of locus functions enables accurate categorization through signal ratio comparison while maintaining a manageable level of complexity through clear role assignment
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 simplifies DNA categorization and profiling, increases accuracy, and eliminates the need for external controls, allowing for cost-effective identification of DNA sources and types, including natural versus artificial DNA, with high reliability.
Implementation Method 1
digesting a DNA sample with a methylation-sensitive and/or methylation-dependent restriction endonuclease
Implementation Method 2
performing PCR on the digested DNA amplifying at least two genomic loci
Data Source
Figure 1(A)~1
Figure 2
Figure 3(A)~3(D)
AI summary
The present application describes methods for accurate and cost-effective categorization of DNA samples into different types of in vitro generated DNA or different types of natural DNA such as from different tissues and/or physiological/pathological states. The invention achieves categorization by comparing "signal ratios" that are correlated to ratios of methylation levels at specific genomic loci, but does not rely on calculation of actual methylation levels at any genomic locus. Therefore the disclosed inventive method eliminates the requirement for external DNA species and controls, thereby simplifying and increasing the accuracy of the assay. The described inventive technology also enables performing the categorization of DNA together with DNA profiling in the same reaction, thereby allowing for concomitant categorization and determination of identity of the samples.