Stem-Loop Adapter Primers for LAMP Nucleic Acid Amplification
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Solution Overview
Problem
Existing nucleic acid amplification methods, particularly for short-chain nucleic acids like fragmented DNA and miRNA, suffer from insufficient sensitivity and specificity, especially when using blood or body fluids as specimens, and often involve complex procedures.
Innovation Solution
A nucleic acid amplification method using a Bw adapter primer with a stem-loop structure and an Fw adapter nucleotide with a 3'-end extension-inhibiting modification to form a dumbbell-shaped template, which is then amplified using the LAMP method, simplifying and enhancing specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used for amplifying short-chain nucleic acids, then amplification can be achieved, but detection sensitivity and specificity are insufficient
Solution Approach 1:
The invention changes the structural parameters of the template nucleic acid by introducing stem-loop structures at both ends through adapter primers. This structural modification enables the LAMP method to specifically recognize and amplify the target sequence, dramatically improving detection sensitivity and specificity for short-chain nucleic acids while maintaining amplification reliability
Solution Approach 2:
The invention uses adapter primers as intermediaries to bridge the gap between conventional PCR and LAMP methods. These adapter primers add stem-loop structures to the amplified product, transforming it into a template suitable for LAMP amplification, thereby enabling high-sensitivity detection of short-chain nucleic acids
2Measurement precision
If LAMP method is used with linear template structure, then amplification can be performed, but detection sensitivity and specificity remain insufficient
Solution Approach 1:
The invention changes the structural parameters of the template from linear to dumbbell-shaped with stem-loop structures at both ends. This structural transformation enables the LAMP method to achieve high detection sensitivity and specificity by providing unique recognition sites for the primers, while the complexity is managed through standardized adapter primer designs
3Measurement precision
If stem-loop structure is added to template for LAMP amplification, then detection sensitivity improves, but the number of steps increases and simplicity decreases
Solution Approach 1:
The invention merges the adapter primer addition step with the initial PCR amplification step. The adapter primers are incorporated during the first amplification round, and the resulting product with stem-loop structures automatically serves as the template for LAMP amplification in the same reaction system, eliminating separate processing steps and maintaining operational simplicity
Solution Approach 2:
The adapter primers designed in the invention enable the amplified product to self-form stem-loop structures that serve as perfect templates for LAMP amplification. The system is designed so that the amplification process itself generates the required template structure, eliminating the need for additional processing 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 method allows for simple and specific amplification of short-chain nucleic acids, reducing non-specific amplification and eliminating the need for complex temperature and cycle control, thereby improving detection sensitivity and specificity.
Implementation Method 1
a Bw adapter primer having a stem-loop structure and an Fw adapter nucleotide having a stem-loop structure
Implementation Method 2
synthesizing a complementary strand of the target region of the target nucleic acid by a reverse transcription reaction
Implementation Method 3
synthesizing a complementary strand starting from a 3'-end of the Bw adapter primer by an extension reaction
Data Source
Figure 1
Figure 2A(a)~2A(b)
Figure 2B(a)~2B(b)
AI summary
The present invention provides a nucleic acid amplification method including: step A including annealing a Bw adapter primer comprising structure (a): 5'-B1c-BL-B1-N3c-3' containing an annealing region N3c, a loop region BL, and stem regions B1c and B1 and a target region of a target nucleic acid, synthesizing a base sequence complementary to a base sequence on a 5'-end side of the target region starting from a 3'-end of the Bw adapter primer, and obtaining a first template nucleic acid in which a stem-loop structure is added to a 5'-end of a complementary strand of the target region; step B including annealing an Fw adapter nucleotide comprising structure (c): 5'-F1c-FL-F1-N5'-3' containing an annealing region N5', a loop region FL, and stem regions F1c and F1; and having an extension-inhibiting modification at the 3'-end and the first template nucleic acid, synthesizing a complementary strand of the Fw adapter nucleotide starting from a 3'-end of the first template nucleic acid, and obtaining a second template nucleic acid comprising structure (d): 5'-B1c-BL-B1-N3c-N5c-F1c-FLc-F1-3' in which a stem-loop structure is added to a 3'-end of the first template nucleic acid; and step C including amplifying base sequence Nc comprising N3c and N5c of the second template nucleic acid by a LAMP method by using an Fw inner primer comprising structure (e): 5'-F1c-F2-3' and a Bw inner primer comprising structure (f): 5'-B1c-B2-3' with the second template nucleic acid as a template.