Uracil-Intolerant Polymerase for Reducing Methylation False Positives
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Solution Overview
Problem
Existing methods using cytosine deaminases result in false positive detection of 5-methylcytosine (5mC) and/or 5-hydroxymethylcytosine (5hmC) due to the deamination of unmethylated cytosines, leading to inaccurate sequencing results.
Innovation Solution
Utilizing uracil-intolerant polymerases like KAPA HiFi, Ultra II Q5, or Phusion HiFi, and/or uracil DNA glycosylase (UDG) to remove uracil residues from DNA fragments, followed by PCR amplification to prevent false positive detections.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cytosine deaminase is used to deaminate methylated cytosines, then 5mC and 5hmC can be detected, but false positive detection occurs due to deamination of unmethylated cytosines
Solution Approach 1:
The patent removes uracil residues (the harmful byproduct of cytosine deamination) from the DNA library fragments using uracil-intolerant polymerases and/or UDG treatment. This extraction of the harmful uracil component eliminates false positive detections while preserving the ability to detect true 5mC and 5hmC sites through selective deamination.
Solution Approach 2:
The patent introduces uracil-intolerant polymerases and UDG as intermediary agents that specifically recognize and remove uracil residues. These intermediaries act as filters between the cytosine deaminase step and the sequencing step, allowing true methylation signals to pass while blocking false positive uracil signals.
2Reliability
If uracil-intolerant polymerase is used to remove uracil residues, then false positive detection is reduced, but additional processing steps are required
Solution Approach 1:
The patent combines multiple functions into the PCR amplification step by using uracil-intolerant polymerases that simultaneously perform DNA synthesis and uracil residue removal. This merging of functions reduces the need for separate treatment steps while maintaining false positive reduction.
Solution Approach 2:
The uracil-intolerant polymerase serves multiple functions: it acts as a DNA polymerase for amplification and simultaneously functions as a uracil removal mechanism. This multi-functionality reduces overall process complexity by eliminating the need for separate uracil removal and amplification steps.
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
Reduces false positive detection of 5mC and 5hmC by ensuring DNA fragments are uracil-free, thereby improving the accuracy of sequencing assays.
Implementation Method 1
subjecting the sample to at least one round of second strand synthesis by contacting the sample including DNA library fragments with an uracil-intolerant polymerase
Implementation Method 2
contacting the sample including DNA library fragments with an uracil DNA glycosylase (UDG) and an endonuclease resulting in DNA library fragments cleaved at uracil residues
Implementation Method 3
a cytosine deaminase has deaminated methylated cytosines
Implementation Method 4
subjecting the sample of double stranded DNA uracil-free library fragments to polymerase chain reaction (PCR) amplification
Data Source
AI summary
Described herein are methods of removing false positive uracils due to the deamination of unmethylated cytosines in assays using engineered cytosine deaminases to deaminate methylated cytosines, the methods utilizing enzymes that discriminate against uracil residues, such as for example, uracil-intolerant polymerases, uracil DNA glycosylase (UDG), and/or USER™ (Uracil-Specific Excision Reagent) enzyme, to remove false positive uracil residues from cytidine deaminase mediated methylation sequencing assays.