DNA Methylation Analysis With Carrier DNA and Unique-End Tagging
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Solution Overview
Problem
Existing methods for analyzing DNA methylation in low amounts, such as from a single cell, suffer from significant DNA loss and damage during chemical or enzymatic conversion, and transposition methods result in a 50% loss of target nucleic acid due to transposon sequence tagging, making it unsuitable for rare or precious samples.
Innovation Solution
The use of transposomes with unique and/or different priming site sequences to fragment and tag genomic DNA, reducing loss by ensuring each fragment has distinct barcode sequences at its ends, and employing carrier DNA to protect the sample during conversion while allowing separation and amplification of targeted DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chemical or enzymatic conversion is performed to analyze DNA methylation, then methylation detection capability is improved, but DNA loss and damage increase up to 90%
Solution Approach 1:
The patent introduces carrier DNA as an intermediary substance that absorbs the harmful effects of chemical conversion reagents (such as bisulfite). The carrier DNA serves as a sacrificial target that protects the precious sample DNA from degradation during the conversion process, thereby enabling methylation analysis while minimizing DNA loss.
Solution Approach 2:
The patent applies beforehand cushioning by pre-adding carrier DNA to the conversion reaction mixture before the actual chemical conversion begins. This carrier DNA is present in advance to cushion or buffer the harsh conversion conditions, protecting the sample DNA from direct exposure to damaging reagents and reducing DNA loss.
2Productivity
If in vitro transposition is used to amplify single-cell genomes, then amplification efficiency is improved, but 50% loss of target nucleic acid occurs due to transposon sequence tagging
Solution Approach 1:
The patent extracts or removes the problematic transposon sequence tags from the amplification process. Instead of using traditional transposition methods that require tagging with transposon sequences (causing 50% loss), the invention employs a tag-free amplification approach that directly amplifies the sample DNA without requiring the addition of external transposon sequences, thereby eliminating the associated DNA loss.
3Loss of substance
If carrier DNA is added to reduce DNA loss during conversion, then DNA protection is improved, but sample mixture cannot be later distinguished
Solution Approach 1:
The patent applies local quality by making the carrier DNA and sample DNA distinguishable through localized differences. The carrier DNA is treated differently (e.g., different labeling, different fragmentation patterns, or different amplification conditions) compared to the sample DNA, allowing the two to be differentiated in the final mixture despite both being present in the same reaction.
Solution Approach 2:
The patent uses segmentation to differentiate carrier DNA from sample DNA by introducing unique molecular identifiers or barcodes to the sample DNA. This segmentation allows computational distinction between carrier and sample sequences during data analysis, enabling the sample information to be extracted from the mixed population.
Data Source
AI summary
The present disclosure provides a method for methylation analysis of genomic fragments.