Allele-Specific Reactive Primer for High-Fidelity Mutation Detection

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Solution Overview

Problem

Conventional allele-specific PCR methods face challenges with DNA polymerases having proofreading activity, as mismatched bases at the 3' end of primers can lead to non-specific amplification due to proofreading activity, limiting their use in detecting mutations.

Innovation Solution

The use of allele-specific reactive primers (ASRP) with modified nucleotides at the 3' end, which are designed to be removed by proofreading activity when non-complementary, ensuring specific amplification of target nucleic acids in the presence of DNA polymerases with proofreading activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If DNA polymerase with proofreading activity is used in allele-specific PCR, then high fidelity in DNA replication is improved, but non-specific amplification occurs due to removal of mismatched bases at the 3' end

Engineering Contradiction:

Improvefidelity in DNA replicationVSAvoidnon-specific amplification

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The invention modifies the chemical parameter of the primer's 3' end by incorporating non-natural nucleotide analogs (such as LNA, 2'-O-methyl RNA, or phosphorothioate modifications) that alter the primer's interaction with the DNA polymerase's proofreading activity, allowing the primer to resist excision even when mismatched, thereby preventing non-specific amplification while maintaining high fidelity replication

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If conventional allele-specific primers are used with DNA polymerase having proofreading activity, then the primer can bind to target DNA, but the mismatched base at the 3' end is removed leading to loss of allele-specificity

Engineering Contradiction:

Improveprimer binding to target DNAVSAvoidallele-specificity

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The invention uses a disposable modified nucleotide at the 3' end of the primer that is designed to be consumed or altered during the first PCR cycle, creating a permanent mark that prevents re-binding and non-specific amplification in subsequent cycles, thereby maintaining allele-specificity throughout the amplification process

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Measurement precision

If the 3' end of primers is labeled or modified to prevent proofreading removal, then allele-specificity is maintained, but the label may interfere with PCR efficiency or detection

Engineering Contradiction:

Improveallele-specificityVSAvoidPCR amplification efficiency

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The invention introduces an intermediary modified nucleotide (such as LNA or 2'-O-methyl RNA) at the 3' end of the primer that acts as a mediator between the natural DNA template and the DNA polymerase, allowing the primer to maintain hybridization stability and resist proofreading removal without significantly interfering with the polymerase's ability to extend the primer and amplify the target sequence

Inventive Principle:

Principle #24Intermediary (Mediator)

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

This approach allows for precise detection and amplification of target nucleic acids, including single nucleotide polymorphisms and methylation status, by preventing non-specific amplification and ensuring that the primer only acts when complementary, enhancing the specificity and accuracy of mutation detection.

Implementation Method 1

DNA polymerase with 3'->5' proofreading activity ensures high fidelity in DNA replication

Methodology Applied

Scientific Effect3' exonuclease activity: Enzyme

Implementation Method 2

an allele-specific reactive primer (ASRP) which comprises i) a nucleotide sequence complementary to the target nucleic acid and ii) one or more modified nucleotide(s) located in a region from a nucleotide located right before of a non-complementary nucleotide to 5' direction of the primer

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentEP2982762B1Nucleic acid amplification method using allele-specific reactive primer

Publication Date: 2018.08.01 GENOMICTREE

Figure 1A~1D
Figure 2~3
Figure 4A~4B

AI summary

The present invention relates to a nucleic acid amplification method using an allele-specific reactive primer (ASRP) which is designed to solve problems of conventional allele-specific PCR. More specifically, the present invention relates to a detection method of a target nucleic acid, comprising a DNA polymerase having proofreading activity and a base sequence complementary to the target nucleic acid, wherein the target nucleic acid is amplified in the presence of an ASRP which is modified in such a manner that one or more bases from the base immediately in front, in the 5'-end direction, of a base that is cut off by the proofreading activity of the DNA polymerase when a non-complementary base exists at the 3' end, to the base at the 5' end, cannot serve as a primer for polymerization. The detection method using an ASRP according to the present invention is a technique with very high specificity of amplification due to the characteristics of the ASRP and the proofreading DNA polymerase, and is capable of effectively detecting mutations (point mutation, insertion, and deletion) including single nucleotide polymorphism (SNP). Furthermore, the method can also be used for detecting the presence of CpG methylation after bisulfite treatment, or for amplifying and detecting a target DNA starting with a desired base sequence from a DNA library.