Modified Polymerases for Bisulphite Modified DNA Amplification
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Solution Overview
Problem
Current methods for bisulphite modification of nucleic acids result in significant DNA degradation, limiting the analysis of methylation status in small cell samples or ancient specimens, and are unable to amplify large genomic regions due to polymerase inefficiencies with abasic sites and bulky adducts.
Innovation Solution
Development of modified enzymes, such as thermophilic or mesophilic polymerases, reverse transcriptases, and endonucleases, capable of efficiently processing bisulphite-modified nucleic acids, including those with abasic sites and sulphonate groups, without the need for desulphonation, allowing for improved PCR amplification and reverse transcription.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard polymerases are used for PCR amplification of bisulphite modified DNA, then amplification can be performed, but the polymerases stall at abasic sites and cannot efficiently amplify DNA containing bulky adducts such as sulphonate groups
Solution Approach 1:
The patent modifies the polymerase enzyme through site-directed mutagenesis to change its biochemical parameters. Specifically, mutations are introduced to alter the polymerase's active site to accommodate and process abasic sites and bulky adducts that would normally cause stalling, thereby enabling reliable amplification of bisulphite modified DNA
Solution Approach 2:
The patent introduces a modified polymerase as an intermediary enzyme that bridges the gap between bisulphite modified DNA templates and successful amplification. This engineered polymerase acts as a mediator that can process the modified nucleic acids without stalling, enabling the PCR reaction to proceed through problematic regions
2Object-affected harmful factors
If desulphonation is performed at high temperatures in alkaline medium to remove sulphonate groups, then bulky adducts are removed, but the majority of nucleic acid damage and loss occurs during this procedure
Solution Approach 1:
The patent extracts or removes the problematic desulphonation step from the bisulphite modification protocol. By using a modified polymerase that can process DNA with retained sulphonate groups, the procedure eliminates the high-temperature alkaline treatment that causes extensive nucleic acid degradation, thereby preserving the integrity of the template
Solution Approach 2:
The patent skips the traditional desulphonation step entirely in the PCR amplification protocol. The modified polymerase is capable of amplifying DNA that retains the sulphonate groups from bisulphite modification, allowing the procedure to rush through to amplification without the damaging intermediate desulphonation step
3Reliability
If small fragments of DNA are amplified after bisulphite treatment, then amplification can be successful, but large genomic regions cannot be analyzed due to polymerase inefficiencies
Solution Approach 1:
The patent modifies the polymerase enzyme's parameters through mutagenesis to enhance its processivity and ability to handle bisulphite modified DNA. These parameter changes enable the polymerase to maintain amplification efficiency over longer DNA fragments, extending the usable range from small fragments to large genomic regions including intact genes
4Quantity of substance
If the complete genome is sequenced to determine genome-wide methylation profile, then comprehensive methylation analysis is achieved, but the huge number of gaps in sequence prevents successful assembly
Solution Approach 1:
The patent modifies the polymerase to process bisulphite modified DNA more efficiently, enabling longer and more continuous sequence reads. This parameter change in enzyme performance reduces the number of gaps in genome assembly by successfully amplifying and sequencing larger contiguous regions of the genome
Solution Approach 2:
The patent creates an asymmetric approach where the polymerase is specifically engineered to handle the asymmetric damage pattern introduced by bisulphite modification (abasic sites and sulphonate groups at specific positions), allowing comprehensive genome coverage while maintaining assembly precision through targeted enzyme modification
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
These enzymes enhance PCR amplification efficiency and enable the analysis of methylation states in small cell samples and large genomic regions, maintaining higher molecular weight DNA and reducing nucleic acid loss, thereby overcoming the limitations of existing methods.
Implementation Method 1
use of an enzyme for copying or amplifying bisulphite modified or treated nucleic acids
Data Source
AI summary
The invention relates to the use of enzymes for copying or amplifying bisulphite modified or treated nucleic acids, wherein the enzymes are more effective in copying or amplifying the nucleic acid compared with native Taq polymerase under substantially the same conditions.