Purified Polymerase-Template Complexes for High-Density Sequencing
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Solution Overview
Problem
Current single molecule nucleic acid sequencing methods face challenges in achieving high throughput and yield due to low fractions of active polymerase-enzyme complexes and inefficient use of observation volumes.
Innovation Solution
A method involving the production of a reaction mixture with polymerase enzyme complexes, extension of complementary strands, and purification to enhance the fraction of active complexes within a desired size range, followed by loading onto substrates like zero mode waveguides to achieve higher active polymerase densities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If polymerase enzyme complexes are produced using conventional methods, then the reaction mixture can be prepared, but the fraction of active polymerase-enzyme complexes is low
Solution Approach 1:
The patent applies preliminary action by performing a pre-incubation step where polymerase enzymes are allowed to bind to template nucleic acids before the actual sequencing reaction. This pre-formed complex ensures that only active polymerase-enzyme complexes proceed to the sequencing step, thereby increasing the fraction of active complexes and improving overall productivity.
2Measurement precision
If observation volumes are made extremely small for single molecule analysis, then sensitivity and reagent cost improve, but the fraction of observation volumes containing a single active complex decreases
Solution Approach 1:
The patent extracts or separates active polymerase-enzyme complexes from inactive ones through purification steps before loading onto observation volumes. This ensures that each extremely small observation volume receives a high proportion of active complexes, maintaining detection sensitivity while improving the reliability that each volume contains a single active complex.
3Productivity
If polymerase enzymes are immobilized in arrays of small wells, then throughput increases, but the fraction of active complexes in each well remains low
Solution Approach 1:
The patent performs preliminary formation and purification of polymerase-enzyme complexes before distributing them into arrays of small wells. This preliminary action ensures that each well receives pre-validated active complexes, thereby maintaining high throughput while improving the fraction of active complexes per well.
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 increases the fraction of active polymerase-enzyme complexes and optimizes the use of observation volumes, leading to higher throughput and yield in nucleic acid sequencing.
Implementation Method 1
extending a complementary strand such that a portion of the polymerase enzyme complexes have complementary strands within a desired size range
Implementation Method 2
purifying the reaction mixture to enhance the relative amount of polymerase enzyme complex having complementary strands within the desired size range
Data Source
AI summary
Methods, Compositions, and Systems are provided for obtaining polymerase-template complex mixtures with improved levels of active polymerase. In some aspects active polymerase-template complex is separated by affinity for nucleoside phosphate moieties attached to a resin. In some aspects, a polymerase-template complex is exposed to reaction conditions in which a complementary strand to the template is produced. The extended reaction mixture is purified by reaction with a resin comprising nucleoside phosphate moieties. This purified mixture can be loaded onto substrates and can be used for further analyzes including single molecule sequencing.