Split Primer Design for Polymorphic Nucleic Acid Amplification
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Solution Overview
Problem
Current nucleic acid amplification technologies face challenges with highly polymorphic targets, such as the HIV genome, where primer mismatches prevent effective amplification, and require specific GC content regions that can be difficult to find, especially when interrupted by polymorphic diversity or secondary structures.
Innovation Solution
The use of split primers that target multiple unique locations on a nucleic acid template, separated by one to five nucleic acid bases, allowing for amplification even in polymorphic regions and overcoming the need for exact complementarity and optimal GC content.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional primers are used for amplification, then amplification works well for non-polymorphic targets, but amplification fails for highly polymorphic targets like HIV due to primer mismatches
Solution Approach 1:
The primer is divided into multiple separate oligonucleotide segments that can independently hybridize to different regions of the target nucleic acid. This segmentation allows the primer to bind effectively even when individual regions contain polymorphisms, as other segments can still provide stable hybridization. The patent describes primers composed of multiple oligos (e.g., 2-10 oligos) that collectively bind to the target, with each oligo contributing to overall binding stability without requiring perfect complementarity at every position.
2Manufacturing precision
If primers require exact complementarity and optimal GC content, then amplification specificity is high, but primer design becomes difficult when target regions are interrupted by polymorphic diversity or secondary structures
Solution Approach 1:
By dividing the primer into multiple oligonucleotide segments, the design process becomes more flexible. Each segment can be independently optimized for local GC content and secondary structure considerations, while the collective segments provide overall binding stability. This approach eliminates the need to find a single continuous region with optimal properties, as the patent demonstrates that distributed segments can achieve equivalent or superior binding characteristics.
Solution Approach 2:
The patent applies parameter changes by allowing variation in the number, length, and sequence composition of individual oligonucleotide segments. Designers can adjust parameters such as oligo length (e.g., 10-50 nucleotides each), GC content distribution, and spacing between segments to optimize for specific target characteristics including polymorphic regions and secondary structures, while maintaining overall primer functionality.
3Device complexity
If single continuous primers are used, then primer structure is simple, but primer cannot effectively target multiple separated regions on polymorphic nucleic acid
Solution Approach 1:
The primer is segmented into multiple oligonucleotide components that can bind to separate regions on the target nucleic acid. This segmentation enables the primer to effectively target polymorphic regions by distributing binding sites across multiple locations, increasing the probability that at least some segments will find complementary sequences despite polymorphisms. The patent describes primers comprising 2-10 separate oligos that collectively provide the necessary binding affinity and specificity.
Solution Approach 2:
The multi-segment primer design provides universal applicability to diverse and polymorphic targets. By creating a primer system where multiple segments collectively bind to the target, the approach becomes universally applicable to various polymorphic nucleic acids including HIV, influenza, and other variable genomes. Each segment can be designed to target conserved regions, while the collective structure adapts to polymorphic variations.
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 enables efficient amplification of nucleic acid sequences by increasing primer specificity and affinity, reducing false negatives and nonspecific amplification, while accommodating polymorphic diversity and varying GC content, as demonstrated by successful real-time PCR with Dengue virus split primers.
Implementation Method 1
The primers may be, for example, less than 100 bases and may include at least two regions capable of binding to at least two target regions
Implementation Method 2
Methods of extension of a nucleic acid are also known to those skilled in the art
Data Source
AI summary
Disclosed are methods and compositions for use in nucleic acid amplification or extension reactions.