Cleavable Primer Nucleic Acid Library Preparation
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Solution Overview
Problem
Current nucleic acid sequencing methods are hindered by expensive and time-consuming sample preparation, particularly in generating nucleic acid fragments with overhanging bases, which are inefficient and require large amounts of starting material.
Innovation Solution
A method involving the use of cleavable primers and adaptors to generate partially complementary double-stranded nucleic acid fragments with overhangs, allowing for efficient enzymatic and biochemical reactions, such as targeted pairing and ligation, within a single reaction vessel, reducing the need for multiple cleanup steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to generate nucleic acid fragments with overhanging bases, then sequencing libraries can be prepared, but the process is expensive and time-consuming
Solution Approach 1:
The patent combines multiple library preparation steps (end repair, A-tailing, adapter ligation) into a single streamlined protocol using pre-designed adapter oligonucleotides that perform multiple functions simultaneously, reducing both time and cost while maintaining library preparation efficiency
Solution Approach 2:
The patent uses pre-synthesized adapter oligonucleotides with predetermined sequences and structures that are ready for direct use, eliminating the need for time-consuming in-situ synthesis of adapters during the library preparation process
2Quantity of substance
If traditional overhang generation methods are used, then nucleic acid fragments can be prepared for sequencing, but large amounts of starting material are required
Solution Approach 1:
The patent modifies the chemical parameters of the adapter oligonucleotides, incorporating unique molecular identifiers (UMIs) and specific sequence compositions that enable efficient binding and reduction of starting material requirements while maintaining overhang generation efficiency
3Reliability
If multiple cleanup steps are performed during library preparation, then purity can be improved, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent integrates purification functions into the adapter oligonucleotide design itself, where the adapters facilitate both ligation and simultaneous purification in a single step, reducing the number of separate cleanup operations needed while maintaining library purity
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 method enhances the efficiency of library preparation by reducing the amount of starting nucleic acids required and simplifying the process, enabling more efficient ligation and sequencing library generation with fewer cleanup steps.
Implementation Method 1
cleaving the cleavable 5′ ends of the first extension products and/or the second extension products, thereby forming second double-stranded duplexes having one or more overhangs
Implementation Method 2
such overhangs can be annealed to their complementary sequence, allowing the targeted pairing of nucleic acid fragments
Implementation Method 3
extending the first set of random primers to generate first extension products hybridized to the single-stranded polynucleotides
Implementation Method 4
ligating one or more double stranded adapters comprising a 3′ overhang to the second double-stranded duplexes, thereby forming the adaptor-ligated library
Data Source
AI summary
The disclosure provides for single amplification and double amplification methods for preparing nucleic acid samples for sequencing.