PCR Normalization via Stochastic Barcoding for Gene Expression
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Solution Overview
Problem
Current methods for measuring gene expression in single cells face challenges in accurately quantifying low-abundance nucleic acids due to amplification biases in PCR, leading to inaccurate gene expression measurements.
Innovation Solution
The use of stochastic barcoding with unique molecular labels to label mRNA during reverse transcription, followed by PCR amplification and sequencing, allows for the correction of amplification biases and accurate quantification of mRNA molecules by analyzing sequencing data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to increase the amount of nucleic acids for sequencing, then the quantity of nucleic acids is increased, but amplification biases occur leading to inaccurate gene expression measurements
Solution Approach 1:
The patent applies preliminary action by performing stochastic barcoding during reverse transcription before PCR amplification. Unique molecular labels are attached to each mRNA molecule at the earliest possible stage, creating a permanent record of the original molecule count that survives subsequent amplification steps. This allows accurate quantification even after PCR introduces amplification biases.
Solution Approach 2:
The patent uses unique molecular labels (barcodes) as intermediaries between the original mRNA molecules and the final sequencing data. These labels serve as a mediator that captures the initial molecule count information and transmits it through the PCR amplification process without being affected by amplification biases, enabling accurate reconstruction of original nucleic acid quantities.
2Measurement precision
If stochastic barcoding with unique molecular labels is used to correct amplification biases, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The patent merges the barcoding function with the reverse transcription process by incorporating unique molecular labels directly into the reverse transcription step. This combination eliminates the need for separate barcoding steps and integrates multiple functions (reverse transcription, cDNA synthesis, and molecular labeling) into a single streamlined process, reducing overall system complexity.
Solution Approach 2:
The patent creates universal barcoding sequences that can be applied to all mRNA molecules regardless of their specific sequence or abundance level. These universal labels serve multiple functions: identifying individual molecules, enabling PCR amplification, and providing sequencing handles. This multi-functionality reduces the need for multiple specialized reagents and simplifies the overall system design.
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 digital counting of mRNA molecules, correcting for amplification biases and providing reliable gene expression profiles of single cells, even at low abundance levels.
Implementation Method 1
barcoding copies of a first target using a plurality of barcodes to generate copies of a first barcoded target... hybridizing the copies of the first target to the plurality of barcodes
Implementation Method 2
amplifying the copies of the first barcoded target, using at least two first forward primers for the first target having different sequences and a first reverse primer for the first target, to generate a first plurality of barcoded first target amplicons
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for PCR normalization. In some embodiments, after barcoding copies of a higher abundance target (e.g., a cDNA species), the barcoded copies are amplified using a pair of forward primers comprising one or more mismatches and a reverse primer. The amplified copies can be further linearly amplified using a forward primer comprising the sequence of one of the pair of forward primers, and a reverse primer.