Circularized DNA Template Preparation for High-Throughput Sequencing
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Solution Overview
Problem
Current methods for preparing nucleic acid templates are time-consuming and expensive, and do not reliably produce high-quality templates suitable for high-throughput DNA sequencing systems, which are essential for efficient and cost-effective genome analysis.
Innovation Solution
The method involves generating overlapping nucleic acid fragments from clonal populations, circularizing them, and distributing these circularized variants into low copy number reaction volumes, using techniques such as single-walled carbon nanotubes, enzymatic digestion, and ligase-mediated circularization, to produce high-quality templates for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional cloning and cell culture methods are used to prepare nucleic acid templates, then the templates can be produced, but the process is time-consuming and expensive
Solution Approach 1:
The invention extracts and eliminates the time-consuming cloning and cell culture steps from the traditional template preparation workflow. By using in vitro transcription and direct amplification methods, the patent removes the biological cloning phase entirely, achieving template preparation without the associated time delays and costs.
Solution Approach 2:
The patent performs preliminary actions by pre-synthesizing RNA templates and preparing amplification-ready nucleic acid sequences before the actual sequencing reaction. This allows templates to be ready for immediate use in high-throughput sequencing without requiring time-consuming cloning procedures.
2Reliability
If conventional nucleic acid purification protocols are used, then nucleic acid templates can be produced, but they are not sufficiently free of sequencing reaction inhibitors
Solution Approach 1:
The invention uses in vitro transcription to create RNA templates and subsequent amplification to generate multiple copies of the nucleic acid sequence of interest. This copying process produces clean, amplified templates that are free from the contaminants present in conventional purification protocols, as the synthesis occurs in a controlled in vitro environment rather than through extraction from complex biological samples.
Solution Approach 2:
The patent replaces mechanical purification protocols (which involve multiple extraction, precipitation, and washing steps that can leave residual contaminants) with a biochemical synthesis approach. By using enzymatic in vitro transcription and amplification, the system generates high-purity templates directly, eliminating the need for lengthy purification procedures that struggle to remove all inhibitors.
3Quantity of substance
If conventional methods are scaled to quantities useful for high throughput sequencing, then more templates can be produced, but the problems of time consumption and contamination are magnified
Solution Approach 1:
The invention merges multiple functions into a single in vitro system that simultaneously performs template synthesis, amplification, and purification. By combining in vitro transcription, PCR amplification, and cleanup into an integrated workflow, the patent achieves both high quantity and high efficiency, producing sufficient templates for high-throughput sequencing without the time consumption that would be magnified by scaling conventional methods.
Solution Approach 2:
The patent utilizes parameter changes in the form of exponential amplification through PCR, where the quantity of templates increases dramatically with each cycle. This allows the system to produce the required quantities of high-quality templates rapidly, avoiding the linear scaling limitations of conventional methods where increasing quantity would proportionally increase time and contamination risk.
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 reduces nucleotide misincorporation errors and enables the rapid, efficient production of high-quality nucleic acid templates, facilitating faster and cheaper high-throughput DNA sequencing by eliminating the need for laborious cloning and cell culture techniques.
Implementation Method 1
binding the template nucleic acid to a plurality of single-walled carbon nanotubes (CNTs) of similar length
Implementation Method 2
ligase-mediated circularization
Data Source
AI summary
Provided are methods of producing low copy number circularized nucleic acid variants that can be distributed to reaction volumes. The methods include providing a template nucleic acid; producing a population of clonal nucleic acids from the template nucleic acid; generating a set of partially overlapping nucleic acid fragments from the population of clonal nucleic acids; circularizing the partially overlapping nucleic acid fragments to produce circularized nucleic acid variants; and aliquotting the circularized nucleic acid variants into reaction volumes. Related compositions of nucleic acid templates are also provided.