Duplex Primer Design for Uniform Nucleic Acid Amplification
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Solution Overview
Problem
Existing nucleic acid amplification methods using random primers result in a wide range of amplicon sizes and require multiple steps, making them inefficient and resource-intensive, while also lacking the ability to incorporate unique tail sequences for easy detection or sequencing post-amplification.
Innovation Solution
The use of duplex primers with a first strand having a target-binding region and a unique tag sequence on its 5′-terminus, and a second strand complementary to the tag sequence, which anneals preferentially to the 3′-terminus of the target nucleic acid, allowing for the generation of longer, uniform amplicons with tags at both ends, thereby simplifying post-amplification processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If random primers are used for amplification, then coverage of multiple target sequences is improved, but amplicon size uniformity deteriorates
Solution Approach 1:
The primer design incorporates a specific structural feature (hairpin loop or duplex configuration) that creates different functional regions within the same primer. The 3′-terminal region binds to target sequences with random specificity for broad coverage, while the 5′-terminal region with complementary sequence forms a stable hairpin or duplex structure that directs annealing preferentially to the 3′-terminus of targets, ensuring uniform amplicon sizes.
2Adaptability or versatility
If multiple amplification steps are used, then unique tail sequences can be incorporated, but amplification time and resource consumption increase
Solution Approach 1:
The invention combines multiple functions into a single primer structure. The hairpin loop or duplex primer simultaneously serves as the binding element for target sequences and as the source of unique tail sequences. The complementary sequence at the 5′-terminus that forms the hairpin or duplex structure also becomes incorporated into the amplicon as a unique tail, eliminating the need for separate tailing reactions.
Solution Approach 2:
The primer structure is designed to perform multiple functions: (1) binding to target sequences through the random nucleotide sequence at the 3′-terminus, (2) directing preferential annealing to 3′-termini through hairpin or duplex formation, (3) serving as a template for polymerase extension, and (4) providing unique tail sequences for downstream applications. This multi-functionality reduces the number of separate reagents and steps required.
3Ease of operation
If standard primers are used, then amplification simplicity is maintained, but post-amplification detection and sequencing require additional steps
Solution Approach 1:
The unique tail sequences are incorporated into the primers before the amplification reaction begins. The complementary sequence at the 5′-terminus of the primer forms a hairpin loop or duplex structure that is directly incorporated into the amplified product. This preliminary incorporation of detection/sequencing tags eliminates the need for post-amplification labeling or adapter ligation steps.
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 reduces the number of amplification steps, generates longer and more uniform amplicons with incorporated tags, facilitating easier detection, sequencing, and reducing resource consumption, while allowing for the incorporation of unique tail sequences for enhanced analysis.
Implementation Method 1
The random nucleotide sequence of the duplex primer is annealed to the target nucleic acid. This occurs primarily at the 3′-terminus of the target nucleic acid, which is favored due to base stacking between the 3′-terminus of the target nucleic acid and the 5′-terminus of the second strand of the duplex primer.
Implementation Method 2
Heat denaturation may be used to separate the strands.
Data Source
AI summary
The present invention provides methods of amplifying a target nucleic acid utilizing duplex primer. The first strand of the primer comprises a random nucleotide sequence of about 6 to about 9 nucleotides in length that is able to hybridize to the target nucleic acid and a tag sequence. The second strand of the primer comprises a sequence complementary to the tag sequence allowing the primer to form a duplex and the ability to bind the tag sequence of the product nucleic acid for further amplification. The resulting nucleic acid produced contains tag sequences on both the 3′- and 5′-termini.