Nucleic Acid Sequencing via Multi-Protein Fluorescence Region Selection

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Solution Overview

Problem

Current methods for nucleic acid analysis in biological samples, particularly in heterogeneous cell populations, lack accuracy and objectivity in identifying regions of interest for sequencing, relying on simple histological staining or single protein immunofluorescence, which limits the detailed information obtainable from trace samples.

Innovation Solution

A method that involves generating fluorescent images of biological samples using immunofluorescence detection of multiple target proteins, selecting regions of interest based on protein expression patterns, and removing subsamples for nucleotide sequence analysis, utilizing techniques like laser microdissection and advanced sequencing methods to provide comprehensive protein and DNA information from homogeneous subsections.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of operation

If simple histological staining or single protein immunofluorescence is used for region selection, then the procedure is simple and fast, but the identification accuracy and objectivity of regions of interest deteriorates

Engineering Contradiction:

Improvesimplicity of region selection procedureVSAvoidaccuracy of region identification

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The procedure is segmented into distinct stages: initial simple staining for rapid overview, followed by targeted immunofluorescence staining of specific proteins of interest. This segmentation allows the workflow to benefit from both simplicity (in the initial screening) and precision (in the targeted analysis of specific regions), resolving the contradiction between ease of operation and measurement precision.

Inventive Principle:

Principle #1Segmentation

2Loss of information

If multiple proteins are analyzed by immunofluorescence, then the information content and accuracy of region identification improves, but the procedure complexity and time consumption increases

Engineering Contradiction:

Improveinformation content from sampleVSAvoidcomplexity of immunofluorescence procedure

Core Design Contradiction:

Loss of informationVSDevice complexity

Solution Approach 1:

The method performs preliminary action by first identifying regions of interest using a subset of proteins or markers, then focuses subsequent detailed analysis only on those selected regions. This preliminary filtering reduces the overall complexity and time required, as not all samples require full multi-protein analysis, thereby resolving the contradiction between information content and procedure complexity.

Inventive Principle:

Principle #10Preliminary action

3Loss of time

If manual identification of regions is used, then the procedure is quick, but the objectivity and reproducibility of region selection deteriorates

Engineering Contradiction:

Improvetime for region selectionVSAvoidobjectivity of region identification

Core Design Contradiction:

Loss of timeVSReliability

Solution Approach 1:

The system incorporates feedback mechanisms where imaging data from immunofluorescence staining is automatically analyzed using image processing algorithms. These algorithms provide objective criteria for region selection based on fluorescence intensity patterns, reducing subjective bias while maintaining efficiency. The feedback loop between imaging, analysis, and selection ensures both objectivity and reproducibility without excessive time loss.

Inventive Principle:

Principle #23Feedback

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

Enables accurate identification and analysis of specific cell subpopulations, mutation detection, and improved diagnostic and prognostic insights by providing detailed protein expression and DNA sequence information from a single homogeneous subsection of a heterogeneous sample.

Implementation Method 1

removing a subsample from the region of interest

Methodology Applied

Scientific EffectLaser ablation: Laser Ablation

Implementation Method 2

generating a fluorescent image of the sample by a protocol comprising immunofluorescence detection

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS10597697B2Method of determining a nucleotide sequence of a nucleic acid segment from a region of interest in a biological sample

Publication Date: 2020.03.24 LEICA MICROSYSTEMS CMS GMBH

AI summary

The invention discloses a method for determining a nucleotide sequence of a nucleic acid segment present in a biological sample, comprising the steps of: a) generating a fluorescent image of the sample by a protocol comprising immunofluorescence detection of at least five different target proteins in the sample; b) selecting a region of interest of the sample by comparing the image to a predetermined criterion; c) removing a subsample from the region of interest, and; d) determining a nucleotide sequence of a nucleic acid segment present in the subsample.