In Situ DNA Barcode Readout in Fixed Cells for SNV Detection

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Solution Overview

Problem

Detecting barcode expression in living cells is challenging due to stochastic silencing, bursty expression, and cell-type dependent promoter activity, and there is a need to accurately identify single nucleotide variations in barcodes.

Innovation Solution

A method involving fixing cells with a fixative, generating barcode molecules, hybridizing them with detection probes and amplifier probes, and using fluorescence imaging to determine barcode sequences in situ. This involves contacting fixed cells with pairs of amplifier probes, where the barcode molecules hybridize with detection probes and amplifier probes, and using fluorescence imaging to identify barcode sequences in situ, and using pairs of amplifier probes, where the barcode molecules are fixed cells, and using fluorescence imaging to determine barcode sequences in situ, and using fluorescence imaging to determine barcode sequences in situ, and using fluorescence imaging to determine barcode sequences in situ, and using fluorescence imaging to determine barcode sequences in situ, and using fluorescence imaging to determine barcode sequences in situ, and using fluorescence imaging to determine barcode sequences in situ.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If barcodes are detected in living cells, then barcode expression can be monitored, but detection is challenged by stochastic silencing, bursty expression, and cell-type dependent promoter activity

Engineering Contradiction:

Improvebarcode detection reliabilityVSAvoiddetection system complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies preliminary action by fixing cells before barcode detection. This fixation step is performed in advance to preserve cellular structures and barcode sequences, eliminating the need for live cell maintenance during detection. The fixation process stabilizes the cellular environment, ensuring reliable barcode detection without the complications of stochastic silencing and bursty expression that affect living cells.

Inventive Principle:

Principle #10Preliminary action

2Measurement precision

If barcode sequences are detected with high precision, then single nucleotide variations can be identified, but the detection method must distinguish single nucleotide differences from background noise

Engineering Contradiction:

Improvebarcode sequence detection precisionVSAvoidsingle nucleotide variation detection difficulty

Core Design Contradiction:

Measurement precisionVSDifficulty of detecting and measuring

Solution Approach 1:

The patent uses probe hybridization as an intermediary mechanism to detect single nucleotide variations. Detection probes are designed to hybridize specifically to complementary barcode sequences, with the hybridization strength providing a measurable signal that distinguishes true matches from mismatches. This intermediary hybridization step amplifies the signal from single nucleotide differences, making them detectable against background noise.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Stability of the object's composition

If cells are fixed to preserve spatial relationships, then in situ barcode detection is enabled, but cell viability is lost

Engineering Contradiction:

Improvecellular structure stabilityVSAvoidbarcode expression reliability

Core Design Contradiction:

Stability of the object's compositionVSReliability

Solution Approach 1:

The patent uses copying by generating multiple copies of barcode sequences through in situ transcription after fixation. Phage RNA polymerases are introduced to transcribe the fixed barcode DNA into RNA, creating amplifiable copies that can be detected with high sensitivity. This copying mechanism ensures that even though cells are fixed and original barcode expression is halted, sufficient barcode copies are generated for reliable detection and single nucleotide variation identification.

Inventive Principle:

Principle #26Copying

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

The method enables accurate detection of barcode sequences in fixed cells, allowing for the identification of single nucleotide variations and determination of cell lineages and spatial relationships.

Implementation Method 1

fixing the plurality of cells using a fixative to generate a plurality of fixed cells

Methodology Applied

Scientific EffectFixation:

Implementation Method 2

each of the plurality of barcode molecules comprising the barcode sequence of the barcode polynucleotide in the fixed cell hybridizes to a detection probe

Methodology Applied

Scientific EffectHybridization:

Implementation Method 3

detecting the fluorophore, or fluorescence thereof, of the pair of amplifier probes

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS12516373B2In situ readout of DNA barcodes

Publication Date: 2026.01.06 CALIFORNIA INST OF TECH

AI summary

Disclosed herein include systems, methods, compositions, and kits for in situ readout of barcodes, such as DNA barcodes. Barcode constructs containing a promoter (e.g., a phage promoter) that is inactive in live cells can be integrated in the genomes of cells. Cells can be fixed, and phage RNA polymerase can be used for transcription of the barcode to RNA transcripts. The RNA transcripts can be detected using, for example, fluorescent imaging and used to determine barcode sequences.