Spanning Primer Nucleic Acid Amplification Specificity
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Solution Overview
Problem
Current methods for amplifying target nucleic acids are inefficient in distinguishing between ligated and unligated nucleic acids, leading to reduced specificity and increased generation of spurious amplification products.
Innovation Solution
The use of spanning primers that anneal to both the 3′ and 5′ ends of the target nucleic acid sequence, or to the ligation products formed, to facilitate specific amplification over unligated nucleic acids, thereby enhancing the efficiency and specificity of the amplification process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional amplification methods are used, then amplification of target nucleic acids is achieved, but specificity is reduced and spurious amplification products are generated
Solution Approach 1:
The patent performs ligation of probes to the target nucleic acid sequence before amplification. This preliminary ligation step creates a unique structure that the spanning primer can specifically recognize and bind to, ensuring that only correctly ligated products are amplified. The ligation step prepares the target in advance with a specific structure that enables subsequent specific amplification.
Solution Approach 2:
The spanning primer acts as an intermediary that bridges the ligated probes. It contains sequences complementary to both the 5' end and 3' end of the ligated product, allowing it to bind across the ligation junction. This intermediary primer enables specific amplification by recognizing the unique structure created by probe ligation, while not binding to unligated probes or non-specific targets.
2Reliability
If spanning primers are used to improve specificity, then amplification of ligated products is favored, but the complexity of the amplification system increases
Solution Approach 1:
The spanning primer performs multiple functions: it serves as a primer for DNA polymerase extension, it spans across the ligation junction to ensure specificity, and it enables amplification of only the correctly ligated product. This multi-functional design consolidates what would otherwise require multiple separate components into a single primer sequence, reducing overall system complexity.
Solution Approach 2:
The patent merges the targeting function and the priming function into a single spanning primer. The primer contains both the sequence necessary to bind to the target (through complementarity to the ligated probes) and the functional elements required for amplification. This merging eliminates the need for separate targeting and amplification components, simplifying the system.
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 significantly improves the amplification of target nucleic acid sequences by favoring the amplification of ligated products over unligated ones, resulting in higher specificity and reduced non-specific amplification products.
Implementation Method 1
a first primer comprising (i) a sequence complementary to the 5' end of the target nucleic acid sequence and (ii) a sequence complementary to the 3' end of the target nucleic acid sequence
Implementation Method 2
a polymerase; subjecting the amplification reaction composition to at least one amplification reaction to form at least one amplification product
Implementation Method 3
forming a ligation reaction composition comprising the sample, and a ligation probe set for each target nucleic acid sequence; forming a first test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another
Data Source
AI summary
Methods and kits for detecting whether target nucleic acid sequences are present and/or quantitating target nucleic acid sequences are provided.