Solid-Phase Nucleic Acid Amplification with Chain-Terminating Nucleotides
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Solution Overview
Problem
Current methods for nucleic acid amplification from small samples, such as single cells or tumor biopsies, face challenges in sensitivity, robustness, and cost-effectiveness, particularly in maintaining high-throughput and low lot-to-lot variation of reagents and experimenter skill levels, and struggle with separating DNA products from oligo DNA-labeled antibodies.
Innovation Solution
A method utilizing chain-terminating nucleotide triphosphates in homopolymer addition reactions on solid-phase carriers, combined with exonuclease I treatment, to stabilize and efficiently amplify cDNA from small samples, reducing by-products and enabling clear separation of amplification products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional TdT method is used for cDNA amplification, then amplification can be performed, but the reaction is highly sensitive to parameter variations (enzyme activity, primer content, reaction time) leading to low reliability
Solution Approach 1:
The patent changes the chemical parameter of the nucleotide triphosphate from normal dNTP to chain-terminating ddNTP. This parameter change fundamentally alters the reaction outcome by preventing excessive elongation and primer by-product formation, thereby stabilizing amplification efficiency across different enzyme activities and reaction conditions without requiring strict parameter control
2Manufacturing precision
If exonuclease I treatment is applied to remove unreacted primers, then primer elimination is achieved, but the ratio between DNA substrate and TdT enzyme changes, reducing amplification efficiency
Solution Approach 1:
The patent performs preliminary action by adding chain-terminating ddNTP during the TdT reaction to prevent excessive elongation and by-product formation before exonuclease I treatment. This preliminary modification of the nucleotide composition ensures that even after exonuclease I removes unreacted primers, sufficient cDNA substrate remains for efficient amplification
3Quantity of substance
If high primer density is used on magnetic bead, then more cDNA can be synthesized, but excessive primer-derived by-products are produced, decreasing amplification efficiency
Solution Approach 1:
The patent converts the harmful effect of high primer density (excessive primer by-products) into a beneficial outcome by using chain-terminating ddNTP. The ddNTP transforms the uncontrolled primer elongation into controlled, limited-length extensions that terminate at specific positions, converting the harmful excessive by-product formation into manageable, separable fragments that do not interfere with subsequent amplification
4Reliability
If long reaction time with TdT is used, then more complete reaction occurs, but excessive elongation of primer by-products occurs, making separation from cDNA difficult
Solution Approach 1:
The patent applies the skipping principle by using chain-terminating ddNTP to rush through the reaction completion quickly. The ddNTP causes immediate termination of primer elongation after incorporating a limited number of nucleotides, allowing the reaction to reach its effective completion point without excessive elongation, thus enabling clear separation between cDNA and primer by-products even with extended reaction times
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 achieves robust, sensitive, and cost-effective cDNA amplification from small samples, independent of experimenter skill and reagent lot variation, and allows for simultaneous protein and mRNA expression analysis, improving therapeutic effect judgment and reducing medical resource wastage.
Implementation Method 1
reaction of nucleotide homopolymer addition to cDNA synthesized on a solid-phase carrier
Implementation Method 2
exonuclease I treatment
Implementation Method 3
chain-terminating reaction with a chain-terminating nucleotide triphosphate which is a chain-terminating CTP or a chain-terminating GTP
Data Source
Figure 1
Figure 2
Figure 3~4
AI summary
A nucleic acid amplification method using a solid-phase carrier according to the present invention comprises: capturing a target nucleic acid comprising mRNA on a solid-phase carrier; carrying out complementary-strand synthesis on the solid phase; carrying out exonuclease treatment to degrade and remove unreacted target-capturing nucleic acid on the solid phase; and then carrying out mRNA degradation and homopolymer addition by TdT reaction in the presence of a chain-terminating nucleotide triphosphate. According to the method of the present invention, cDNA can be stably and highly efficiently amplified even from a small amount of sample even in cases where the ratio of the amount of enzyme to the DNA substrate on the solid phase is excessive, where the reaction time is excessive, and/or where reagents show lot-to-lot variation. Further, the amplification method of the present invention can broaden the range of applications of techniques in which analysis using a specific-binding molecule labeled with an oligonucleic acid such as a DNA-labeled antibody and analysis of transcripts are carried out simultaneously.