Sequencing of complete integration loci.

To verify the genomic origin of the integration sequences obtained by LAM-PCR, a direct genomic primer walking from 5′ to 3′ was performed by repeated application of the LAM-PCR direct genomic sequencing protocol in different directions. 5′ Fusion: the 5′ genomic DNA/5′ LTR-cDNA fusion sequences were obtained by initial LAM-PCR reactions. Integration Site: an amplification product spanning the complete 5′ and 3′ integration locus was produced by the second LAM-PCR reaction for each of the analyzed clones. All bands obtained were isolated, purified, and sequenced. In each of the 3 clones analyzed (shown: 2 clones), this second LAM-PCR reaction obtained a sequence that was identical to the sequence obtained in the first LAM-PCR reaction from the 5′ end of the amplification product to the 5′ genomic proviral fusion sequence (arrows), then identical to the 3′ integration flank to the 3′ end of the amplification product. 3′ Fusion: the 3′ LTR-cDNA/3′ genomic DNA fusion sequences were obtained by conventional PCR sequencing. Together with a 3′ LTR U5 forward primer (LTR), the 3′ flanking genomic primer pairs (3′ Fl) produced a clone-specific amplification product by nested amplification on 1 μg of transduced genomic DNA from day 339 peripheral blood granulocytes. Sequencing confirmed that the amplification products consisted of genomic 3′ flanking sequence, the 4-bp repeat identical to the 4 bp flanking the 5′ LTR U3 of the respective clone and the expected 3′ LTR U5 vector sequence. The 4-bp genomic sequence direct repeat reduplicated by the retrovirus integrase is enlarged. The first 4 proviral DNA nucleotides are shown in underlined italics. C indicates unrelated DNA controls; LAM1, 5′ LTR extension primer of the initial LAM-PCR; LAM2, 5′ integration site–specific genomic DNA extension primers for the second LAM-PCR based on the 5′ genomic DNA sequences identified in the initial LAM-PCRs.