Cleavable Phosphoramidite Linkers for Bidirectional Sequencing
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in increasing read-length due to the expense of synthesizing nucleoside phosphoramidites with bridged-phosphorothioate internucleotide linkages and are limited to sequencing in one direction.
Innovation Solution
The development of novel cleavable phosphoramidite linkers that allow chemical removal from nucleic acid sequences, enabling longer read-lengths and bidirectional sequencing by incorporating detectable signals like fluorophores, which are economically synthesized and can be chemically cleaved using agents like AgNO3 and I2.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If bridged-phosphorothioate internucleotide linkages are used to increase read-length, then read-length is improved, but synthesis cost and complexity increase due to commercially unavailable nucleoside phosphoramidites
Solution Approach 1:
The patent employs a disposable, chemically cleavable linker that is inexpensive to synthesize and can be easily removed after use. This linker replaces the need for expensive, specialized bridged-phosphorothioate linkers, achieving the same functional outcome (enabling longer read-length sequencing) at lower cost and with simpler synthesis procedures.
Solution Approach 2:
The patent modifies the chemical structure of the linker by incorporating a chemically cleavable phosphoramidite group that can be synthesized using standard, commercially available reagents. This parameter change in the linker's chemical composition allows for easier, cheaper synthesis while maintaining the ability to extend read-length and enable bidirectional sequencing.
2Length of moving object
If bridged-phosphorothioate linkages are used to extend read-length, then read-length is improved, but sequencing directionality is limited to one direction
Solution Approach 1:
The chemically cleavable linker is designed to serve multiple functions: it enables extension of read-length, allows for bidirectional sequencing (both 5'-3' and 3'-5' directions), and can be removed chemically after use. This multi-functional design replaces the need for separate specialized linkers for different sequencing directions, achieving both extended read-length and bidirectional capability with a single versatile component.
3Adaptability or versatility
If chemically cleavable linkers are used to enable bidirectional sequencing and longer read-length, then sequencing versatility is improved, but additional chemical cleavage steps are required
Solution Approach 1:
The linker is designed to be self-cleaving through a simple chemical reaction with AgNO3 or I2, eliminating the need for complex enzymatic or mechanical removal processes. The chemical cleavage step is straightforward and can be performed easily, making the added complexity of bidirectional sequencing and extended read-length manageable through a simple, well-defined chemical process.
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 doubles the read-length of nucleic acid sequencing while allowing sequencing in both directions, overcoming the limitations of existing technologies and reducing synthesis costs.
Implementation Method 1
chemically cleaving the cleavable linker to remove the detectable label
Data Source
AI summary
Linkers and methods for determining a nucleotide sequence of a reference oligonucleotide are provided.