FFPE DNA Preparation Using Nuclease to Remove Sequencing Artifacts
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Solution Overview
Problem
Formalin-fixed paraffin-embedded (FFPE) tissue samples contain formaldehyde-induced crosslinks, DNA fragmentations, and DNA lesions that cause false-positives in DNA sequencing results, leading to inaccurate cancer diagnosis due to mismatched DNA pairs.
Innovation Solution
A method involving the use of a nuclease, such as Mung Bean Nuclease, to digest mismatched DNA pairs in FFPE samples before analysis, followed by treatments with uracil-DNA glycosylase and thymine DNA glycosylase to remove mismatched DNA bases, improving DNA quality for Next Generation Sequencing (NGS) analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If FFPE tissue samples are used for DNA analysis, then sample storage and transport is simplified, but DNA artifacts and false-positives increase
Solution Approach 1:
The patent applies preliminary action by treating DNA with uracil-DNA glycosylase and thymine DNA glycosylase before sequencing to remove deaminated bases (uracil and thymine lesions) that cause false-positives. This pre-treatment eliminates artifacts before they interfere with accurate mutation detection, allowing reliable analysis of FFPE samples that would otherwise be unsuitable.
2Reliability
If UDG treatment is applied to remove uracil lesions, then C:G>T:A changes due to uracil are reduced, but thymine lesions remain undetected
Solution Approach 1:
The patent merges the functions of two different glycosylases: uracil-DNA glycosylase (UDG) to remove uracil lesions and thymine DNA glycosylase (TDG) to remove thymine lesions. By combining these two enzymatic treatments, the method achieves comprehensive removal of both types of deamination artifacts, making the approach versatile against multiple DNA damage types rather than targeting only one.
3Reliability
If multiple enzymatic treatments are applied to remove DNA artifacts, then sequencing accuracy improves, but process complexity increases
Solution Approach 1:
The patent creates a universal DNA preparation protocol that handles multiple types of DNA damage (uracil lesions, thymine lesions, and other artifacts) through a standardized multi-enzyme treatment approach. This universal method can be applied to all FFPE samples regardless of their specific degradation patterns, simplifying the overall workflow despite the multiple enzymatic steps by providing a single comprehensive solution rather than requiring separate protocols for different damage types.
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
The method enhances the accuracy of DNA sequencing by reducing background noise from artificially mutated DNA, thereby improving the reliability of cancer diagnosis and treatment recommendations based on genomic information.
Implementation Method 1
contacting the tissue sample with a nuclease that digests mismatched DNA pairs in the DNA
Implementation Method 2
Treatment of FFPE DNA with uracil-DNA glycosylase (UDG) before PCR-based sequencing can reduce apparent C:G>T:A single-nucleotide changes due to uracil lesions
Implementation Method 3
it was suggested that thymine lesions may be removed using the repair enzymes thymine DNA glycosylase (TDG)
Data Source
AI summary
The disclosed technology relates to a method of analyzing DNA from a biological sample. In one aspect, the method comprises obtaining a sample a sample from a subject, the sample comprising DNA that comprises double-stranded DNA (dsDNA) suspected of having one or more thymine lesions formed by deamination of 5-methylcytosines, wherein the thymine lesions form base pair mismatches in the dsDNA, contacting the sample with a nuclease that digests dsDNA at base pair mismatches; harvesting DNA from the nuclease-treated sample; and performing an analysis on undigested DNA harvested from the nuclease-treated sample.