Oligonucleotide Density Control for Nucleic Acid Amplification
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Solution Overview
Problem
Current nucleic acid amplification methods require multiple sample titration steps to achieve optimal cluster density on solid supports, which can lead to inefficiencies, reagent waste, and increased processing time, as cluster density is often dependent on the concentration of the nucleic acid sample.
Innovation Solution
A method involving immobilized capture and amplification oligonucleotides on a solid support, where capture oligonucleotides are immobilized at a lower density than amplification oligonucleotides, allowing for selective hybridization and extension of template polynucleotides, thereby controlling cluster density independently of the sample concentration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple sample titration steps are performed to achieve optimal cluster density, then cluster density control is improved, but processing time and reagent consumption increase
Solution Approach 1:
The capture oligonucleotides are pre-immobilized on the solid support at a controlled lower density before sample application. This preliminary arrangement of capture oligonucleotides establishes the maximum potential cluster density in advance, eliminating the need for subsequent titration steps to determine optimal density. The system is prepared with the correct capture capacity before the sample is introduced, thereby reducing processing time while maintaining precise cluster density control.
2Manufacturing precision
If multiple sample titration steps are performed to achieve optimal cluster density, then cluster density control is improved, but reagent consumption increases
Solution Approach 1:
The capture oligonucleotides are pre-immobilized on the solid support at a controlled lower density before sample application. This preliminary arrangement of capture oligonucleotides establishes the maximum potential cluster density in advance, eliminating the need for subsequent titration steps to determine optimal density. The system is prepared with the correct capture capacity before the sample is introduced, thereby reducing processing time while maintaining precise cluster density control.
3Adaptability or versatility
If capture oligonucleotides are immobilized at lower density than amplification oligonucleotides, then cluster density is controlled independently of sample concentration, but oligonucleotide immobilization complexity increases
Solution Approach 1:
The oligonucleotide population is segmented into two distinct functional groups: capture oligonucleotides and amplification oligonucleotides. Capture oligonucleotides are immobilized at a lower density and are responsible for selectively binding template polynucleotides, while amplification oligonucleotides are immobilized at a higher density and facilitate subsequent amplification. This segmentation allows independent control of cluster density through capture oligonucleotide density, making the system adaptable to varying sample concentrations without requiring complex real-time adjustments.
4Reliability
If capture oligonucleotides are used to control cluster density, then reproducibility across multiple chips is improved, but device complexity increases
Solution Approach 1:
The density parameter of capture oligonucleotides is changed and controlled during immobilization to be lower than that of amplification oligonucleotides. By establishing a fixed, controlled density of capture oligonucleotides on the solid support, the system achieves consistent and reproducible cluster density across multiple chips. This parameter control eliminates variability introduced by sample concentration differences, thereby improving reliability and reproducibility of results across different experimental runs and chips.
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 eliminates the need for precise sample titration, ensures consistent cluster density across multiple chips, reduces reagent usage, and enhances reproducibility by controlling cluster formation through the ratio of capture to amplification oligonucleotides, rather than sample concentration.
Implementation Method 1
a plurality of capture oligonucleotides each having a different sequence capable of hybridising to a selected region of the nucleic acid sample
Implementation Method 2
extending the capture oligonucleotides to generate extension products complementary to the template polynucleotides
Data Source
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AI summary
The invention provides methods for controlling the density of different molecular species on the surface of a solid support. A first mixture of different molecular species is attached to a solid support under conditions to attach each species at a desired density, thereby producing a derivatized support having attached capture molecules. The derivatized support is treated with a second mixture of different molecular species, wherein different molecular species in the second mixture bind specifically to the different capture molecules attached to the solid support. One or more of the capture molecules can be reversibly modified such that the capture molecules have a different activity before and after the second mixture of molecular species are attached. In particular embodiments, the different molecular species are nucleic acids that are reversibly modified to have different activity in an amplification reaction.