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- A. Integration of the retroviral vector U3neoSV1 into
genomic DNA. Vector U3neoSVfs is a derivative of U3neoSV1, and is identical
to it except for insertion of a single T near the 5' end of the neo
gene within the LTR. In this example integration is in an exon.
- B. Inverse PCR and sequencing of insertion site. The
diagram shows the vector integrated in genomic DNA. The provirus contains
two LTR sequences (big squares) in tandem and about 4 kb internal DNA.
The genomic DNA/vector junctions are amplified by inverse PCR: the genomic
DNA is digested with NspI, then ligated to produce DNA circles, and
amplified by PCR using as primers either neoIN10 + neoIN1 (at left;
called NL), or neoIN22 + neoIN21 (at right; called NR). The NspI restriction
enzyme recognition site used for inverse PCR is located in the two copies
of the Neo gene. The internal 4.5 kb proviral NspI fragment is too large
to be amplified under our PCR reaction conditions. The sequencing primers
neoES and neoLTR3 are used to sequence the junctions. By convention,
we have oriented all sequences in our database as the positive strand:
the insertion sites of "NL" and "NR" sequences are at the right and
left end of the sequence respectively.
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