Fluorescent Protein DNA Mapping Without Cleavage Damage
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Solution Overview
Problem
Current DNA sequencing technologies face limitations in short read length and information loss, and conventional analysis methods using sequence-specific restriction enzymes or fluorescent dyes cause DNA cleavage, necessitating the development of a substance that can bind sequence-specifically and fluoresce without causing DNA cleavage.
Innovation Solution
A composition comprising an adenine/thymine (A/T)-specific DNA binding protein linked with a first fluorescent protein and an A/T-non-specific DNA binding protein linked with a second fluorescent protein, allowing for efficient optical identification of single DNA molecules without separate sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If conventional fluorescent dyes (YOYO-1) are used for DNA visualization, then fluorescence signal is obtained, but DNA cleavage is caused due to light-induced damage
Solution Approach 1:
The patent introduces fluorescent proteins (GFP, RFP, YFP, BFP) as intermediary substances that bind to DNA and provide fluorescence signals without causing light-induced DNA cleavage. These fluorescent proteins serve as mediators between the detection system and DNA, eliminating the harmful effects of conventional dyes while maintaining the beneficial fluorescence signal for optical mapping
Solution Approach 2:
The patent employs restriction enzymes as disposable tools that cleave DNA at specific sequences to generate fragment patterns for identification. These enzymes perform their function once and are then discarded, enabling sequence-specific analysis without requiring the DNA to withstand repeated harsh treatment that would cause damage
2Measurement precision
If sequence-specific restriction enzymes are used for DNA analysis, then sequence-specific patterns are obtained, but DNA cleavage occurs
Solution Approach 1:
The patent uses fluorescent proteins as intermediaries that bind to DNA and provide sequence-specific signals through their binding characteristics, avoiding the need for enzymatic cleavage. The fluorescent proteins mediate the interaction between detection systems and DNA sequences, enabling precise identification without generating harmful cleavage byproducts
Solution Approach 2:
The patent replaces the mechanical/enzymatic cleavage system with an optical detection system based on fluorescent protein binding. Instead of using restriction enzymes to physically cut DNA at specific sequences, the system uses fluorescent proteins that bind to DNA and provide optical signals, substituting a mechanical process with an optical one that avoids DNA damage
3Measurement precision
If current sequencing technology is used, then nucleotide sequences are obtained, but read length is limited and information is lost in large genomes
Solution Approach 1:
The patent segments large genomic DNA into smaller fragments through controlled cleavage or natural breaking, then uses fluorescent proteins to label and visualize these segments. By analyzing the pattern, size, and arrangement of multiple segments rather than attempting to sequence the entire long DNA molecule continuously, the system overcomes read length limitations while maintaining information about the original large genome structure
Solution Approach 2:
The patent creates optical copies or maps of DNA sequences through fluorescent protein binding patterns. Instead of directly reading the nucleotide sequence with limited read length, the system creates a visual copy or representation of the DNA structure using fluorescent signals, allowing indirect analysis of long genomic regions that exceeds the direct sequencing read length capability
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 composition enables sequence-specific DNA mapping and analysis at a single-DNA molecule level, facilitating chromosomal organization studies and detecting chemically modified or damaged DNA, with improved accuracy and efficiency.
Implementation Method 1
an adenine/thymine (A/T)-specific DNA binding protein linked with a first fluorescent protein; and an A/T-non-specific DNA binding protein linked with a second fluorescent protein. The first fluorescent protein and the second fluorescent protein may exhibit different colors differentiated from each other.
Data Source
AI summary
The present invention relates to a composition for DNA sequence analysis and a method for DNA sequence analysis, the method comprising treating a sample with the composition. The composition of the present invention can attain efficient optical identification at a single-DNA molecule level by linking both an A/T-specific DNA-binder agent and an A/T-non-specific complementary DNA-binder agent to DNA, and thus can be helpfully used in studying chromosomal organization of genomes, protein immunolocalization, and the like.