Single-Cell Multiomics Using PTA Amplification for Accurate Sequencing
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Solution Overview
Problem
Current methods for nucleic acid amplification and sequencing from small samples, especially single cells, lack scalability, accuracy, and efficiency, particularly in simultaneous analysis of RNA, DNA, and proteins.
Innovation Solution
A method involving isolating a single cell to amplify RNA by RT-PCR, generating a cDNA library, and using terminator nucleotides to terminate genomic DNA replication, followed by sequencing both libraries, with optional dUTP and uracil-tolerant polymerases to enhance purity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid amplification methods are used for single cell analysis, then sequencing can be performed, but accuracy and scalability are insufficient
Solution Approach 1:
The patent segments the amplification process into distinct phases: initial amplification using polymerase, termination using terminator nucleotides, and selective removal using uracil-tolerant polymerase. This segmentation allows each step to be optimized independently, achieving both high accuracy through controlled amplification and scalability through automated termination and purification steps.
Solution Approach 2:
The patent introduces an intermediary mechanism using uracil-tolerant polymerase as a mediator between the amplification step and the sequencing step. This intermediary enzyme selectively removes terminator nucleotides and purifies the amplified DNA, enabling accurate sequencing while maintaining scalability through a standardized purification process.
2Quantity of substance
If RNA is amplified from single cells, then transcriptome information is obtained, but genomic DNA contamination affects purity
Solution Approach 1:
The patent extracts and removes genomic DNA contamination from the cDNA library through selective purification steps. By using uracil-tolerant polymerase that specifically recognizes and removes terminator nucleotides, the method extracts unwanted genomic DNA sequences while preserving the desired cDNA, thereby improving cDNA purity without compromising yield.
Solution Approach 2:
The patent changes the chemical parameter of the nucleotide mixture by incorporating terminator nucleotides with specific properties (e.g., uracil bases) that can be selectively recognized and removed. This parameter change enables differential treatment of cDNA and genomic DNA during purification, achieving high purity while maintaining adequate yield.
3Productivity
If terminator nucleotides are used to stop replication, then sequencing can be performed, but incomplete termination reduces accuracy
Solution Approach 1:
The patent implements a feedback mechanism where uracil-tolerant polymerase continuously monitors and removes remaining terminator nucleotides from the amplified DNA. This feedback loop ensures complete termination of replication and thorough removal of terminators, achieving both high sequencing efficiency and accurate sequence data without contamination from incomplete termination.
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 achieves highly accurate and scalable nucleic acid amplification and sequencing, improving sequence representation, uniformity, and accuracy, enabling comprehensive multiomic analysis of single cells, including proteins, DNA, and RNA.
Implementation Method 1
contacting the genomic DNA with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide which terminates nucleic acid replication by the polymerase
Implementation Method 2
amplifying the RNA by RT-PCR to generate a cDNA library
Implementation Method 3
contacting the cDNA library with an enzyme configured to digest or remove polynucleotides from the genomic DNA library
Data Source
AI summary
Provided herein are compositions and methods for accurate and scalable single cell multiomics methods, and their applications for mutational analysis in research. diagnostics, and treatment. Further provided herein are multiomics methods for parallel analysis of DNA, RNA, and/or proteins from single cells using Primary Template-Directed Amplification (PTA) nucleic acid amplification.