Solid Support Nucleic Acid Amplification Method
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Solution Overview
Problem
Current nucleic acid purification and amplification methods are slow, tedious, expensive, difficult to automate, and lack sensitivity, specificity, precision, and accuracy, making them unsuitable for diverse applications, particularly in next-generation sequencing.
Innovation Solution
A method involving first and second oligonucleotides affixed to a solid support, where the first oligonucleotide is blocked to prevent extension and complementary to a target nucleic acid, and the second oligonucleotide is identical to a portion of the target, allowing for the immobilization and amplification of specific nucleic acids through primer extension and polymerase action, with optional use of tag sequences and displacer/blocker oligonucleotides for increased specificity and control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nucleic acid purification and amplification methods are used, then the process can be performed with standard reagents and equipment, but the method is slow, tedious, expensive, and difficult to automate
Solution Approach 1:
The amplification process is divided into distinct functional regions: a solid support with immobilized capture oligonucleotides, reagent reservoirs containing primers and enzymes, and defined reaction zones. This segmentation allows parallel processing of multiple targets simultaneously while maintaining standardized操作流程, thereby increasing productivity without proportionally increasing overall system complexity.
Solution Approach 2:
Solid support particles serve as intermediaries that concentrate and organize all necessary amplification components (template nucleic acids, capture oligonucleotides, primers, enzymes) in defined spatial locations. This intermediary structure enables automated liquid handling systems to efficiently deliver reagents while maintaining reaction integrity, resolving the contradiction between automation capability and method complexity.
2Measurement precision
If traditional amplification methods are used, then standard reagents can be employed, but sensitivity, specificity, precision, and accuracy are insufficient
Solution Approach 1:
The solid support particles create localized microenvironments where amplification reactions occur in highly concentrated zones. Capture oligonucleotides are site-specifically immobilized to ensure precise target capture, and reagent delivery is localized to reaction zones containing templates. This local quality enhancement improves detection accuracy and specificity without requiring proportional increases in total reagent quantities.
Solution Approach 2:
The invention transitions from traditional two-dimensional (surface well) amplification to three-dimensional amplification within suspended or surface-bound particles. This dimensional change increases the effective concentration of reactants and enables more precise spatial control of molecular interactions, thereby improving measurement precision while maintaining reagent efficiency.
3Reliability
If multiple purification steps are performed, then nucleic acid quality can be improved, but the process time and labor requirements increase
Solution Approach 1:
Capture oligonucleotides are pre-immobilized on solid support particles before sample addition, creating ready-to-use amplification stations. This preliminary preparation eliminates the need for post-capture purification steps, as the solid support structure inherently separates bound targets from unbound contaminants during subsequent washing operations, thereby maintaining nucleic acid quality while reducing overall process time.
Solution Approach 2:
The invention combines target capture, amplification initiation, and product enrichment into a single integrated solid-phase process. Multiple traditional purification functions (target isolation, concentration, and enrichment) are merged into the solid support-based amplification workflow, eliminating sequential purification steps and reducing both process time and labor requirements while maintaining product quality.
4Extent of automation
If manual processing steps are used, then flexibility in protocol adjustment is maintained, but automation and manufacturing scalability are limited
Solution Approach 1:
The solid support particles serve multiple functions: target capture, reaction vessel, and product carrier. This multi-functionality enables a single standardized particle type to be used across different assay configurations and automation platforms, facilitating automation implementation without sacrificing operational flexibility. Standardized particles can be processed by various automated liquid handling systems while maintaining protocol adaptability.
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 enables rapid, efficient, and accurate immobilization and amplification of nucleic acids, improving sensitivity and specificity, and allowing for directional amplification and multiplexing, which is essential for precise sequencing and gene expression analysis.
Implementation Method 1
The first oligonucleotide is blocked to prevent extension from the 3'-terminus and has a sequence complementary to a first portion of a target nucleic acid. A sample is applied to the solid support and target sequences in the sample bind the first oligonucleotide.
Implementation Method 2
A primer containing a target-binding region and a polymerase promoter sequence is hybridized to the bound target nucleic acid.
Implementation Method 3
The primer is extended by polymerase to produce a first duplex nucleic acid. The second oligonucleotide is extended by polymerase to produce a second duplex nucleic acid containing a first nucleic acid and a second nucleic acid. Multiple copies of a third nucleic acid are then generated by adding polymerase specific to the promoter sequence to the solid support thereby amplifying the target nucleic acid.
Data Source
Figure 1A
Figure 1A
Figure 1B
AI summary
The present invention provides methods for amplifying a nucleic acid from a mixture of nucleic acids utilizing a solid support. Methods are provided utilizing user-defined primer oligonucleotides for directional amplification. Methods are also provided utilizing blocker and displacer oligonucleotides for generating amplified target nucleic acids of defined length. A sample is applied to the solid support and the target nucleic acid within the sample binds a first oligonucleotide affixed to a solid support. A primer sequence containing a target binding region and a polymerase promoter sequence is annealed to the bound target nucleic acid and extended producing a first duplex nucleic acid. The target sequence is removed leaving a first nucleic acid that binds a second oligonucleotide affixed to a solid support. The second oligonucleotide is extended to produce a second duplex nucleic acid that contains a second nucleic acid. The second nucleic acid is amplified by adding a polymerase.