Modified Nucleotide Signatures for cfDNA Identification

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Solution Overview

Problem

Current methods for analyzing short nucleic acid fragments face challenges in creating unique signatures for identification, especially in large genomes like humans, and fail to preserve epigenetic modifications during amplification, requiring a more efficient and reliable method for identifying and quantifying these fragments with minimal starting material.

Innovation Solution

The method involves using modified nucleotides during replication to generate a unique signature for short nucleic acid molecules, allowing for their identification and detection of epigenetic modifications without interfering with native modifications, using a polymerase with modified nucleotide triphosphates and specific antibodies for detection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If amplification is performed to increase the amount of starting material, then the quantity of nucleic acid is improved, but epigenetic modifications are lost and amplification efficiency varies

Engineering Contradiction:

Improvequantity of nucleic acidVSAvoidpreservation of epigenetic modifications

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The invention extracts only the necessary information (sequence and epigenetic modifications) directly from single nucleic acid molecules without performing amplification. By using single-molecule sequencing technology, the method obtains sufficient data from individual molecules, eliminating the need for amplification that would otherwise cause loss of epigenetic modifications.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention creates multiple copies of the sequencing signal through repeated imaging and detection of the same single nucleic acid molecule. By capturing multiple images during the sequencing process and combining the signals, the method achieves sufficient quantity of data without amplifying the original nucleic acid, thus preserving epigenetic modifications.

Inventive Principle:

Principle #26Copying

2Measurement precision

If small oligonucleotides are used to create barcodes for identification, then the identification capability is improved, but the method fails for short nucleic acid fragments where binding density is insufficient

Engineering Contradiction:

Improveidentification capabilityVSAvoididentification of short fragments

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The invention segments the nucleic acid molecule into individual nucleotides for sequential detection. By imaging each nucleotide position separately during synthesis and using modified bases with distinct imaging signals, the method creates identification capability at the single-nucleotide level, which is particularly effective for short fragments where overall barcode density would be insufficient.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention applies different imaging properties to different nucleotide positions along the molecule. Each modified base incorporates specific imaging dyes or labels that emit distinct signals, creating local quality variations that enable precise identification of each position. This local differentiation provides robust identification even for short fragments with limited total positions.

Inventive Principle:

Principle #3Local quality

3Measurement precision

If modified nucleotides are used to generate unique signatures, then the identification accuracy is improved, but the complexity of the replication reaction increases

Engineering Contradiction:

Improveidentification accuracyVSAvoidcomplexity of replication reaction

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The invention changes the chemical parameters of the nucleotides by incorporating modified bases with unique imaging properties. These parameter changes enable direct optical detection of each nucleotide position during synthesis, providing high identification accuracy. The modified nucleotides are designed to maintain compatibility with standard polymerase enzymes, minimizing the increase in reaction complexity.

Inventive Principle:

Principle #35Parameter changes

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 the accurate identification and quantification of short nucleic acid fragments with minimal reagents, preserving epigenetic information and providing a unique signature for each molecule, improving the reliability and efficiency of nucleic acid analysis.

Implementation Method 1

replicating the nucleic acid molecule with a polymerase in the presence of a modified nucleotide triphosphate

Methodology Applied

Scientific EffectPolymerization:

Implementation Method 2

detecting the position of the modified nucleotide incorporated in the newly synthesised nucleic acid molecule

Methodology Applied

Scientific EffectAntibody binding:

Data Source

PatentUS20250003011A1Sample preparation methods using modified bases for rapid identification and sequencing

Publication Date: 2025.01.02 DEPIXUS

AI summary

The present invention provides a method for preparing a nucleic acid sample for producing hairpin molecules, preferably from small dsDNA molecules, notably from cfDNA, or from small RNA molecules such as fragmented RNA molecules, in order to identify them rapidly on nucleic acid analysis instruments (e.g., the MAGNA platform) with a minimal set of reagents.