Cleavable Oligonucleotide Barcodes for Bead-Free PCR Cell Capture
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Solution Overview
Problem
Current methods face challenges in efficiently amplifying poly(A) mRNA molecules during PCR due to the presence of magnetic beads, and there is a need for systems and methods that can quantitatively analyze protein expression while simultaneously measuring gene expression in cells.
Innovation Solution
A method involving the use of cellular component-binding reagents with specific oligonucleotides that bind to cellular components, followed by partitioning cells, hybridizing with oligonucleotide barcodes, extending and separating these components to generate barcoded nucleic acid molecules, and employing cleaving agents to separate and obtain sequence information for protein and gene expression analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If magnetic beads are used to co-localize cells with barcoded reagents, then cell-specific barcoding is achieved, but PCR amplification efficiency is inhibited
Solution Approach 1:
The patent extracts and removes magnetic beads from the PCR reaction mixture after initial cell capture and barcoding. This separation eliminates the inhibitory effect of magnetic beads on PCR amplification while preserving the cell-specific barcoding that was established during the magnetic bead-mediated capture step
Solution Approach 2:
The patent divides the workflow into distinct phases: (1) cell capture and barcoding using magnetic beads, (2) removal of magnetic beads, and (3) PCR amplification without magnetic beads. This segmentation allows each step to be optimized independently, resolving the contradiction between reliable barcoding and efficient amplification
2Measurement precision
If current methods are used to measure gene expression, then mRNA quantification is achieved, but simultaneous protein expression analysis is not possible
Solution Approach 1:
The patent employs universal oligonucleotide sequences and barcoding strategies that can simultaneously track both mRNA and protein molecules. The same barcoded reagents used for mRNA capture can be used for protein capture, enabling simultaneous multi-omics measurement from the same cell population
Solution Approach 2:
The patent merges gene expression analysis and protein expression analysis into a single integrated workflow. Both mRNA and protein are captured, barcoded, and analyzed simultaneously using complementary approaches, allowing correlated measurement of transcriptional and translational states
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 PCR amplification efficiency and allows for simultaneous quantitation of protein and gene expression in cells, overcoming the inhibitory effects of magnetic beads and providing comprehensive expression data.
Implementation Method 1
contacting a plurality of cellular component-binding reagents with a plurality of cells comprising a plurality of cellular component targets and copies of a nucleic acid target... wherein the cellular component-binding reagent specific oligonucleotide comprises DNA
Implementation Method 2
extending the cellular component-binding reagent specific oligonucleotides hybridized to the plurality of oligonucleotide barcodes to generate a plurality of extended cellular component-binding reagent specific oligonucleotides
Implementation Method 3
wherein the cleavage region is 5′ of the first universal sequence and comprises one or more cleavage sites
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets. Some embodiments provide oligonucleotide barcodes comprising a cleavage region. The cleavage region can comprise one or more cleavage sites. In some embodiments, cleaving agents are capable of effectuating cleavage of a phosphodiester backbone of the cleavage region. The oligonucleotide barcode can be associated with a solid support. Methods of separating barcoded nucleic acid molecules from the solid support are also provided in some embodiments.