PCR Primers With Abasic Parts for Template Mutation Tolerance
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Solution Overview
Problem
Conventional PCR methods face challenges in efficiently amplifying different templates with mutated or polymorphic sites, often resulting in non-specific amplification and false-positive results due to primer mismatch and the need for optimized reaction conditions, which increases costs and labor in multiplex PCR diagnostics.
Innovation Solution
The use of primers with abasic parts, such as dSpacer and other nucleosides without specific coding information, inserted at mutated or polymorphic sites within the primer sequences, allowing for the amplification of different templates using a single pair of primers by regulating the melting temperature through the addition of complementary nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional primers are used for PCR amplification, then amplification of target sequences can be achieved, but non-specific amplification and false-positive results occur due to primer mismatch at mutated or polymorphic sites
Solution Approach 1:
The invention changes the chemical parameter of the primer by replacing a nucleotide at the mismatched position with an abasic part (a sugar moiety without a nitrogenous base). This parameter change allows the primer to bind to multiple template variants with different nucleotides at that position, thereby increasing adaptability to different templates while maintaining amplification reliability through the preserved 3'-end complementarity.
2Adaptability or versatility
If annealing temperature is lowered to tolerate mismatches, then amplification of templates with mutations can be achieved, but non-specific amplification increases
Solution Approach 1:
Instead of changing the physical parameter of annealing temperature, the invention changes the chemical structure of the primer by introducing an abasic part. This allows the primer to tolerate nucleotide mismatches at the abasic site position while maintaining specific annealing through the complementary 3'-end region, thus achieving both adaptability and reliability without compromising specificity.
3Adaptability or versatility
If multiple primer sets are used for multiplex PCR to cover different mutations, then comprehensive amplification can be achieved, but device complexity and optimization requirements increase
Solution Approach 1:
The invention creates a universal primer design where a single primer containing an abasic part can function across multiple template variants with different nucleotides at the mutation site. This multi-functional primer replaces the need for multiple specific primers, thereby reducing device complexity and the number of optimization conditions required while maintaining comprehensive coverage of different templates.
4Measurement precision
If primers are designed with high specificity to match exact sequences, then amplification accuracy is improved, but ability to amplify templates with mutations is reduced
Solution Approach 1:
The invention modifies the primer's chemical parameter by substituting a nucleotide with an abasic part at the position corresponding to the polymorphic or mutated site. This parameter change allows the primer to maintain high annealing specificity through the complementary 3'-end while gaining the ability to bind to multiple template sequences with different nucleotides at the abasic site position, thus resolving the trade-off between precision and adaptability.
Data Source
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AI summary
The present invention relates to primers for PCR amplification comprising abasic parts within the primer sequences and a method for PCR amplification using the same. More precisely, the present invention relates to primers capable of amplifying different templates and having abasic parts complementary to mutated site or polymorphic site of template DNA and a method for PCR amplification comprising the steps of mixing the composition for PCR amplification comprising the primers with nucleic acid template; and performing PCR with the mixture. The primers for PCR amplification of the present invention contain abasic parts not having specific coding information in their nucleotide sequences, so that they can amplify different templates having mutated sites at the same time.