Identify the exact site of integration of recombinant lentiviral vectors using the Lenti-X Integration Site Analysis Kit. The replication life cycle of a lentivirus results in integration of a DNA copy of the lentiviral RNA genome into the host genome. The chosen integration site has important consequences for both the expression of your transgene and the phenotype of the host cell. The Lentiviral Integration Site Analysis Kit uses GenomeWalker technology to identify those sites so that they can be sequenced and analyzed.
The GenomeWalker Procedure
- Genomic DNA from transduced clones or populations is digested with blunt-end cutting restriction enzymes.
- The GenomeWalker adaptor is ligated to the blunt ends of the genomic DNA library.
- A primary PCR amplifies those fragments from the library that contain both an adaptor and a lentiviral sequence.
- A secondary ‘nested PCR’ generally produces a single, major PCR product that can be sequenced to determine the integration site. Multiple bands can result from multiple integrations sites and each can be purified and sequenced separately.
Please see data and images section for a schematic of the procedure.
What is in the Kit?
The kit provides all the components and controls needed to isolate and PCR amplify lentiviral integration sites for three cell clones, including:
- NucleoSpin Tissue Kit to purify high quality genomic DNA from transduced mammalian cells
- Three blunt-cutter restriction enzymes, ligase, GenomeWalker adaptor and PCR primers
- Advantage 2 PCR kit for long distance amplification from Genomic DNA
- NucleoSpin Gel and PCR Clean-Up kit for isolating of the amplified band(s)
For the complete list of components, please see the product's Certificate of Analysis and Kit Components List.