Lentivirus Integration Junction Analysis Using Inverse PCR and RCA
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Solution Overview
Problem
Current methods for assessing lentiviral vector delivery and infection in gene therapy lack a quick and accurate method for analyzing the location and copy number of lentivirus provirus integration in the host human genome.
Innovation Solution
A method involving shearing genomic DNA, using inverse PCR with LTR region primers, and rolling circle amplification to analyze the integration junctions, followed by bioinformatic analysis to determine the genomic location and copy number of the lentivirus provirus.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR and sequencing methods are used to analyze lentiviral integration, then the analysis can be performed with standard techniques, but the resolution and accuracy of integration location detection is insufficient
Solution Approach 1:
The method segments the genomic DNA into fragments containing integration junctions by shearing and selective amplification. Inverse PCR amplifies only the regions containing LTR-host genome junctions, separating these from the vast majority of non-junction genomic DNA. This segmentation enables high-resolution analysis of integration sites without requiring analysis of the entire genome, thereby improving detection precision while managing complexity through targeted enrichment.
Solution Approach 2:
The patent introduces rolling circle amplification (RCA) as an intermediary step between conventional PCR and sequencing. RCA amplifies the circularized PCR products containing integration junctions to generate sufficient material for high-precision sequencing analysis. This intermediary amplification step enables accurate detection of integration locations without requiring direct sequencing of low-concentration junction fragments, thus improving measurement precision while using established techniques.
2Measurement precision
If high-resolution analysis of integration sites is performed, then accurate quality assessment of viral transduction is achieved, but the time and resources required for analysis increase
Solution Approach 1:
The method performs preliminary enrichment of integration junction fragments before sequencing through inverse PCR and rolling circle amplification. By pre-concentrating and circularizing only the relevant junction-containing fragments, the method reduces the complexity and time of subsequent sequencing and bioinformatic analysis. This preliminary action ensures that high-resolution analysis can be achieved without proportionally increasing total analysis time, as the enrichment steps are highly efficient and targeted.
3Measurement precision
If the entire genomic DNA is sequenced to find integration sites, then complete coverage is achieved, but the cost and complexity of the analysis increases significantly
Solution Approach 1:
The method extracts and isolates only the specific DNA fragments containing integration junctions from the entire genomic DNA through shearing, selective amplification with LTR-specific primers, and rolling circle amplification. This extraction approach takes out the relevant integration site information from the vast majority of non-relevant genomic DNA, enabling precise identification of integration locations while processing a minimal quantity of DNA, thereby reducing cost and complexity compared to whole-genome sequencing.
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
Provides high-resolution analysis of lentivirus integration sites and copy numbers in the human genome, enabling precise quality assessment of viral transduction.
Implementation Method 1
Multiplying the circularized strands provided with the PCR primers by inverse PCR, thereby obtaining a plurality of linear strands wherein the at least one LTR region and the junction regions are embedded by the PCR primers
Implementation Method 2
Converting the linear strands with embedded LTR and junction regions into circularized and amplification of the circularized strands by RCA into rolonies
Data Source
Figure 1~1(H)
Figure 2~3B
Figure 4
AI summary
The invention is directed to method to obtain the genomic location of a provirus sequence embedded by two LTR regions in a DNA strand