Integration Site Detection via Cyclization and Rolling Circle Amplification
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Solution Overview
Problem
Current methods for detecting the integration site of exogenous genes in host genomes, such as those used in transgenic animals, suffer from low sensitivity, high complexity, and inaccuracies, particularly in detecting low-frequency insertion sites and require substantial sample amounts.
Innovation Solution
A method involving fragmentation, cyclization, rolling circle amplification, and sequencing library construction to accurately determine integration sites, enabling high sensitivity and single-base resolution detection of integration sites using bioinformatics analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional PCR is used to detect integration sites, then the method is simple and convenient to operate, but it has low sensitivity and may produce false positives
Solution Approach 1:
The patent introduces an intermediary step of in vitro transcription to generate RNA templates from the DNA sample. This RNA intermediate serves as a mediator that enhances the detection sensitivity by allowing multiple rounds of reverse transcription and amplification, thereby improving the detection of low-frequency integration sites while maintaining operational simplicity
Solution Approach 2:
The patent changes the physical state parameter of the target nucleic acid by converting DNA to RNA through in vitro transcription. This parameter change enables the use of reverse transcription PCR, which amplifies the signal from low-abundance integration sites, thereby improving detection sensitivity without complicating the overall procedure
2Measurement precision
If Southern Blot is used to detect integration sites, then the sensitivity and accuracy are high, but the operation is complicated and expensive
Solution Approach 1:
The patent extracts and amplifies only the specific integration site regions containing the exogenous gene sequences through targeted PCR amplification after reverse transcription. This extraction of relevant information eliminates the need for complex gel electrophoresis and hybridization steps required by Southern Blot, thereby maintaining high detection accuracy while significantly reducing operational complexity and cost
Solution Approach 2:
The patent uses PCR amplification to create multiple copies of the target integration site sequences from the RNA template. This copying process generates sufficient signal for accurate detection without requiring the complex sample preparation and hybridization procedures of Southern Blot, thus achieving high accuracy with simpler operations
3Measurement precision
If LAM-PCR is used to detect integration sites, then rare insertion sites can be detected, but technical errors are introduced due to restriction enzyme preferences
Solution Approach 1:
The patent replaces the mechanical restriction enzyme digestion step with a template-directed in vitro transcription system. This substitution eliminates the bias introduced by restriction enzyme recognition site preferences, as the RNA template is synthesized based on the actual DNA sequence rather than being dependent on restriction sites. This maintains high sensitivity for detecting rare insertion sites while improving the reliability and accuracy of the results
4Reliability
If DNA hybridization capture is used for rough screening, then false positives are avoided and it is suitable for large numbers of samples, but it cannot accurately locate the exogenous gene
Solution Approach 1:
The patent merges the advantages of hybridization capture (specificity and false positive avoidance) with the location precision of PCR-based methods. By using in vitro transcription to generate RNA templates followed by reverse transcription PCR with gene-specific primers, the method maintains the specificity of hybridization while achieving precise mapping of integration sites through sequence analysis of the amplified products
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 provides accurate and efficient detection of integration sites, including low-frequency insertions, with enhanced sensitivity and reduced experimental time, while requiring less sample material.
Implementation Method 1
subjecting the linear nucleic acid fragments to cyclization to obtain a double-stranded cyclized product
Implementation Method 2
performing rolling circle amplification by taking the double-stranded cyclized product as a template and using a primer specifically bound to the modified nucleic acid
Data Source
AI summary
A method for detecting an integration site. The method comprises: a) subjecting host DNA containing a modified nucleic acid to fragmentation to obtain linear nucleic acid fragments; b) subjecting the linear nucleic acid fragments to cyclization to obtain a double-stranded cyclized product, and then removing uncyclized linear nucleic acid fragments; c) performing rolling circle amplification by taking the double-stranded cyclized product as a template and using a primer specifically bound to the modified nucleic acid; d) subjecting the amplification product to fragmentation and establishing a sequencing library; e) subjecting the sequencing library to on-machine sequencing to obtain a sequencing result; and f) subjecting the sequencing result and an original sequence of the host DNA to alignment to determine the integration site of the modified nucleic acid in the host DNA.