Reaction Site Loading Without Prewetting for Low-Input Sequencing
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Solution Overview
Problem
Existing single molecule analysis techniques require large sample volumes and additional steps like prewetting and washing, which increase the amount of sample needed and compromise loading uniformity and sequencing efficiency.
Innovation Solution
A method involving a loading solution comprising polymerase enzyme complexes, nonionic surfactants, and crowding agents is used to distribute molecules into nanoscale wells without prewetting the surface, reducing sample input by 2- to 10-fold while maintaining sequencing performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional loading methods are used, then molecules can be loaded into reaction sites, but large sample volumes are required and additional prewetting and washing steps are needed
Solution Approach 1:
The patent combines multiple functions into a single loading solution: the solution simultaneously provides polymerase enzyme complexes, nonionic surfactants for surface wetting, and crowding agents for molecular distribution. This unified approach eliminates the need for separate prewetting and washing steps, reducing both sample volume requirements and process complexity while achieving optimal loading into nanoscale wells
Solution Approach 2:
The loading solution acts as an intermediary medium that facilitates the delivery of polymerase enzyme complexes to the reaction sites. The nonionic surfactants in the solution mediate surface wetting and molecular distribution, enabling efficient loading without requiring large sample volumes or multiple processing steps
2Reliability
If prewetting and washing steps are performed, then surface preparation is improved, but sample amount and time are increased
Solution Approach 1:
The loading solution contains nonionic surfactants that perform preliminary surface wetting and preparation actions automatically during the loading process itself. This eliminates the need for separate prewetting and washing steps, reducing loading time while maintaining surface preparation quality through the surfactant-mediated distribution of molecules during the single loading operation
3Manufacturing precision
If conventional loading methods are used, then molecules can be delivered to reaction region, but loading uniformity is compromised
Solution Approach 1:
The patent changes the physical and chemical parameters of the loading medium by incorporating nonionic surfactants and crowding agents. These parameter changes enable improved molecular distribution and loading uniformity across nanoscale wells while reducing the total sample volume required. The surfactants modify surface properties to enhance uniformity, and crowding agents optimize molecular spacing and distribution
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
The method achieves optimal loading with reduced sample input, eliminating prewetting and washing steps, and maintains or improves sequencing efficiency and signal-to-noise ratio.
Implementation Method 1
The loading solution comprises (a) one or more polymerase enzyme complexes comprising a template nucleic acid and a polymerase enzyme; (b) one or more nonionic surfactants; and (c) one or more crowding agents
Implementation Method 2
one or more crowding agents
Data Source
AI summary
Disclosed are methods for directly loading samples to reaction sites on a surface of a substrate without any step of prewetting the surface. The methods result in a reduced sample input amount by at least 2- to 10-fold compared to the conventional protocol that requires the prewetting step.