Double-Stranded Nucleic Acid Sequencing With Separate Strand Primers
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Solution Overview
Problem
Existing nucleic acid sequencing methods for dual strand sequencing are time-consuming and expensive, often requiring synthesis and removal of strands between sequencing reads, which can introduce errors.
Innovation Solution
A method involving hybridization of primers to sense and antisense strands of a nucleic acid concatemer, extending the primers to determine sequences, and optionally incorporating blocking moieties to prevent further extension, allowing sequencing in the presence of both strands without additional synthesis steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If complementary strands are synthesized and/or removed between two sequencing reads, then dual strand sequencing can be performed, but the process becomes cumbersome, time consuming and expensive
Solution Approach 1:
The patent applies preliminary action by pre-synthesizing both sense and antisense strands of the nucleic acid template before sequencing begins. The template is prepared with both complementary strands already in place, eliminating the need for strand synthesis or removal between sequencing reads. This preliminary preparation allows both strands to be sequenced directly without additional time-consuming steps.
2Reliability
If complementary strands are synthesized and/or removed between two sequencing reads, then dual strand sequencing can be performed, but the process becomes cumbersome, time consuming and expensive
Solution Approach 1:
The patent merges the sequencing of both sense and antisense strands into a single continuous process. By having both strands present from the beginning and sequencing them without intermediate strand removal or synthesis, the method combines what would traditionally be separate, complex operations into one streamlined workflow, reducing procedural complexity and potential error sources.
3Productivity
If a strand for second read sequencing is generated from a strand that has been subjected to sequencing, then sequencing can proceed, but errors may be introduced into the sequence results
Solution Approach 1:
The patent segments the sequencing process into two independent parallel pathways: one for sequencing the sense strand and another for sequencing the antisense strand. Each strand is sequenced independently from its own template without using the other strand as a template, preventing error propagation. This segmentation ensures that errors in one sequencing reaction do not affect the other.
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 sequencing time and cost while maintaining accuracy by minimizing amplification cycles and avoiding strand removal, providing high-quality paired sequence data.
Implementation Method 1
hybridizing a primer to the primer binding site in a sequence unit of the antisense strand
Implementation Method 2
extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand
Implementation Method 3
a capping moiety or a blocking moiety can be incorporated into the primer extended along the antisense strand such that the primer is not further extended during step (e)
Data Source
AI summary
A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.