Dendrimer-Based Nucleic Acid Fragmentation for Phasing
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Solution Overview
Problem
Current methods for genomic analysis face challenges in efficiently processing and analyzing nucleic acid samples, particularly in isolating and sequencing nucleic acid fragments from complex biological samples, such as those from single cells or microbiomes, due to limitations in fragment length and phasing accuracy.
Innovation Solution
The use of dendrimers with nucleic acid binding moieties and oligonucleotides, including psoralen and biotin tags, to form complexes with nucleic acids, followed by endonuclease treatment and photocleavable linkage formation, enables the creation of concatemers and barcoded fragments for enhanced sequencing and phasing, allowing for improved analysis of long-range genomic information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used for isolating and sequencing nucleic acid fragments, then the process can be completed, but the fragment length is limited and phasing accuracy is reduced
Solution Approach 1:
The dendrimer is divided into multiple generations (G0-G6) with increasing numbers of binding moieties, allowing progressive fragmentation and phasing of nucleic acid sequences. Each generation provides finer resolution for phasing analysis while maintaining manageable processing complexity through systematic scaling.
Solution Approach 2:
Multiple nucleic acid binding moieties are nested within the dendrimer structure at different generations, enabling simultaneous binding to multiple nucleic acid fragments. This nested arrangement allows complex phasing analysis without proportionally increasing overall system complexity.
2Measurement precision
If higher generation dendrimers with more binding moieties are used, then phasing accuracy improves, but the complexity of the dendrimer structure increases
Solution Approach 1:
The dendrimer structure is segmented into discrete generations (G0-G6), each with a defined number of binding moieties (1-64). This segmentation allows systematic control over complexity while progressively improving phasing accuracy, enabling users to select appropriate generation based on their specific analytical needs.
Solution Approach 2:
The patent systematically varies key parameters including dendrimer generation (G0-G6), number of binding moieties (1-64), and molecular weight (0.5-934 kDa) to optimize the balance between phasing accuracy and structural complexity. This parameter optimization allows tailored solutions for different analytical applications.
3Productivity
If conventional sequencing methods are used, then sequencing can be performed, but sequencing depth is increased and efficiency is reduced
Solution Approach 1:
The dendrimer-based fragmentation method segments nucleic acid molecules into discrete, size-controlled fragments through controlled endonuclease digestion. This segmentation enables more efficient sequencing by reducing the total quantity of sequencing cycles needed while maintaining comprehensive coverage through the systematic fragmentation pattern.
Solution Approach 2:
The dendrimer pre-fragments the nucleic acid molecules before sequencing through controlled endonuclease treatment. This preliminary fragmentation action improves sequencing efficiency by preparing the nucleic acid in an optimized state that requires reduced sequencing depth to achieve adequate coverage and accuracy.
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 enables accurate and efficient genomic analysis, including haplotype phasing and metagenomics, with high phasing accuracy and reduced sequencing depth, facilitating the analysis of complex samples and providing detailed genomic and metagenomic insights.
Implementation Method 1
a dendrimer comprising a plurality of nucleic acid binding moieties
Implementation Method 2
contacting the complex to an endonuclease to cleave the nucleic acid molecule between contact points of the nucleic acid molecule and the plurality of nucleic acid binding moieties
Implementation Method 3
The DBCO modified nucleotides are linked to a biotin azide via a photocleavable linkage
Data Source
AI summary
Provided herein are methods and compositions for nucleic acid processing comprising obtaining a stabilized sample comprising a nucleic acid molecule complexed to at least one nucleic acid binding protein; contacting said stabilized sample to a dendrimer comprising a plurality of nucleic acid binding moieties such that said nucleic acid molecule forms a complex with said plurality of nucleic acid binding moieties; contacting said complex to an endonuclease to cleave said nucleic acid molecule between contact points of said nucleic acid molecule and said plurality of nucleic acid binding moieties creating a plurality of fragments of said nucleic acid molecule each complexed with a nucleic acid binding moiety of said plurality of said nucleic acid binding moieties; isolating said product using an agent that binds to said dendrimer; joining said plurality of fragments to each other to create a concatemer comprising each of said plurality of fragments of said nucleic acid molecule; and isolating said concatemer from said dendrimer.