Transposome Mediated Nucleic Acid Library Preparation
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Solution Overview
Problem
Current methods for preparing nucleic acid samples, such as RNA or transcripts, are time-consuming and often fail to achieve full-length coverage of long nucleic acid molecules, particularly in high-throughput multiplexing applications.
Innovation Solution
The use of a composition comprising a transposome with a polypeptide transposase and a nucleic acid with a mosaic end and a tag, combined with a reverse transcriptase and an oligonucleotide with a mosaic end and a second tag, to generate DNA/RNA hybrid molecules with tags at the 5' ends, enabling efficient fragmentation and tagging in a single reaction, followed by amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nucleic acid sample preparation methods are used, then sequencing libraries can be generated, but the process is time-consuming and requires multiple handling steps
Solution Approach 1:
The patent combines fragmentation and tagging operations into a single transposome-mediated reaction step. The transposome simultaneously fragments the nucleic acid molecules and attaches adapter sequences, eliminating the need for separate fragmentation and adapter ligation steps required by traditional methods, thereby dramatically reducing handling time and increasing productivity
Solution Approach 2:
The transposome serves multiple functions in a single step: it acts as both the fragmentation mechanism and the tagging mechanism. This multi-functional approach consolidates what would traditionally require multiple specialized reagents and steps into one universal reagent system, streamlining the workflow and reducing time loss
2Manufacturing precision
If traditional nucleic acid sample preparation methods are used, then sequencing libraries can be generated, but full-length coverage of long nucleic acid molecules is not achieved
Solution Approach 1:
The transposome is designed with pre-attached adapter sequences that are ready for immediate incorporation during the fragmentation reaction. This preliminary preparation of the tagging mechanism ensures that adapters are efficiently attached during the same step as fragmentation, preventing loss of full-length information that would occur with sequential processing steps
Solution Approach 2:
The invention changes the fundamental parameters of the fragmentation and tagging process by using a single enzymatic reaction instead of multiple physical and chemical steps. This parameter change maintains the integrity of long nucleic acid molecules while achieving both fragmentation and tagging, thereby achieving full-length coverage without increasing procedural complexity
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 simplifies sample preparation, reduces handling time, and achieves near-full-length coverage of nucleic acid molecules, facilitating rapid generation of sequencing libraries for diagnostic, therapeutic, and forensic applications.
Implementation Method 1
Transposase-mediated fragmentation and tagging of nucleic acid molecules can be used in the preparation of nucleic acid molecules
Implementation Method 2
a reverse transcriptase
Data Source
AI summary
Provided herein are methods, compositions, and kits for generating a nucleic acid library. In various embodiments provided herein, transposomes comprising transposases are used in forming dual-end tagged nucleic acid molecules for downstream amplification and nucleic acid molecule processing steps.