Identify the exact site of integration of recombinant retroviral vectors using the Retro-X Integration Site Analysis Kit. The replication life cycle of a retrovirus results in integration of a DNA copy of the retroviral 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 Retroviral Integration Site Analysis Kit uses GenomeWalker technology to identify the integration site(s) 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.
- Primary PCR amplifies those fragments from the library that contain both an adaptor and a retroviral sequence.
- 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 the data and images tab 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 retroviral 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 isolation of the amplified band(s)
For the complete list of components, please see the product's Certificate of Analysis and Kit Components List.