3′-End Phosphorothioate Primers for Linear DNA Amplification
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Solution Overview
Problem
Existing DNA linear amplification methods suffer from non-specific amplification due to primer dimers, off-target binding, and high-fidelity polymerase errors, leading to reduced efficiency and accuracy, especially in fragmented DNA detection.
Innovation Solution
Introduce phosphorothioate modification at the 3′ end of specific primers to prevent ligation with other oligonucleotides and utilize a DNA polymerase with 3′-5′ exonuclease activity to remove mismatched bases, ensuring accurate and specific linear amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If linear amplification is used to avoid base error accumulation and detect fragmented DNA, then detection accuracy for fragmented DNA is improved, but amplification efficiency decreases requiring tens of cycles
Solution Approach 1:
The patent modifies the chemical structure of the primer by introducing phosphorothioate bonds at the 3' end, changing the physical-chemical parameters of the oligonucleotide to prevent non-specific ligation while maintaining specific binding capability, thereby resolving the contradiction between amplification specificity and efficiency
2Productivity
If high concentration of primers is used in multiplex amplification system, then amplification capacity is improved, but primer dimer formation increases causing non-specific amplification
Solution Approach 1:
The patent applies different chemical modifications to different parts of the primer: the 5' end and middle portion maintain normal phosphodiester bonds for stable binding, while the 3' end is modified with phosphorothioate bonds to prevent ligation, creating local quality differences that resolve the contradiction between amplification capacity and non-specific amplification
3Measurement precision
If high-fidelity polymerase with 3'5' exonuclease activity is used to remove mismatched bases, then amplification fidelity is improved, but off-target amplification increases due to removal of mismatched bases at off-target sites
Solution Approach 1:
The patent preemptively blocks the 3' end of the primer with phosphorothioate modification before the polymerase can act, preventing both the beneficial removal of mismatched bases at target sites and the harmful removal at off-target sites, thereby resolving the contradiction between amplification fidelity and off-target amplification
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
Significantly reduces non-specific amplification, enhances the quality of the amplification product, and allows for effective construction of libraries suitable for sequencing, improving the accuracy and efficiency of DNA detection.
Implementation Method 1
a phosphodiester bond of a part of nucleotide backbone at the 3′ end of the specific primer is modified by phosphorothioate
Implementation Method 2
the duality functional group can be removed by a specific enzyme to perform a linear amplification of the specific primer
Data Source
AI summary
A method for amplifying a DNA target region is provided. The method includes: linearly amplifying a fragmented DNA including the target region by a specific primer to provide a linear amplification product, where 3′ end of the specific primer is modified by a duality functional group, a phosphodiester bond of a part of nucleotide backbone at the 3′ end of the specific primer is modified by phosphorothioate, and the duality functional group is used to prevent the 3′ end of the specific primer from ligation with other oligonucleotides and can be removed by a specific enzyme to perform a linear amplification of the specific primer. The method for amplifying a DNA target region effectively reduces a non-specific amplification during the linear amplification by introducing a phosphorothioate modification into the specific primer.