Cre/loxP Nucleic Acid Constructs for HRD Cell Detection
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Solution Overview
Problem
Current methods are inadequate for efficiently detecting DNA homologous recombination deficiency in cells, particularly in cancers like breast cancer, which is crucial for early detection and effective treatment.
Innovation Solution
A set of nucleic acid constructs, comprising a first nucleic acid construct with a cleaved Cre gene and a second nucleic acid construct with loxP sequences and a reporter gene, is used to transfect cells, allowing detection of DNA homologous recombination deficiency through Cre/loxP recombination and reporter gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for DNA homologous recombination deficiency, then detection can be performed, but detection efficiency and accuracy are insufficient
Solution Approach 1:
The patent introduces an intermediary detection system using nucleic acid constructs that mediate between the DNA homologous recombination machinery and the detection signal. The constructs include a donor sequence with homology to the target site, a reporter gene, and selection markers, which serve as intermediaries to amplify and make visible the otherwise invisible molecular repair events, thereby simultaneously improving detection accuracy and efficiency
Solution Approach 2:
The patent replaces conventional mechanical or chemical detection methods with a biological molecular recognition system. The nucleic acid constructs utilize homologous recombination - a natural biological mechanism - to automatically detect and report DNA repair activity through reporter gene expression, substituting complex mechanical detection apparatus with elegant biological molecular interactions that provide both high precision and throughput
2Reliability
If early detection methods are implemented for cancer, then treatment effectiveness can be improved, but current methods lack sufficient sensitivity for early-stage detection
Solution Approach 1:
The patent implements preliminary action by introducing nucleic acid constructs into cells before cancer develops or progresses to advanced stages. The constructs are预先 installed in the cellular system, ready to detect and report DNA repair deficiencies at their earliest manifestations. This preliminary positioning of detection capability enables early cancer detection by capturing molecular signatures of genomic instability before they manifest as overt disease
Solution Approach 2:
The patent employs color changes through reporter genes (such as fluorescent proteins or colorimetric enzymes) that change the optical properties of cells based on DNA repair activity. This visual transformation converts invisible molecular events into detectable color or fluorescence signals, dramatically improving detection sensitivity and enabling reliable early cancer detection through straightforward optical readouts
3Adaptability or versatility
If DNA homologous recombination function is assessed, then drug response prediction is possible, but current assessment methods are too complex and time-consuming
Solution Approach 1:
The patent segments the complex DNA repair assessment into discrete, modular nucleic acid construct units. Each construct is a self-contained module with specific functional elements (promoter, donor sequence, reporter, selection marker) that can independently assess specific aspects of homologous recombination function. This segmentation allows parallel assessment of multiple parameters simultaneously, dramatically reducing assessment time while maintaining comprehensive drug response prediction capability
Solution Approach 2:
The patent uses copying by introducing exogenous nucleic acid constructs that replicate or mimic the endogenous DNA repair process. These constructed systems serve as simplified copies of the complex natural repair machinery, allowing rapid assessment of repair function without needing to directly measure the entire endogenous system. This copying approach enables quick drug response prediction by assessing repair capacity through the engineered proxy system
Data Source
AI summary
According to one embodiment, a set of nucleic acid constructs which detects a DNA homologous recombination deficiency includes a first nucleic acid construct and a second nucleic acid construct. The first nucleic acid construct includes a first promoter sequence and a cleaved Cre gene ligated to downstream of the first promoter sequence. The second nucleic acid construct includes a second promoter sequence, a first loxP sequence ligated to downstream of the second promoter sequence, a reporter gene ligated to downstream of the first loxP sequence, and a second loxP sequence ligated to downstream of the reporter gene.