Gene frequency in PBMC after infusion of transduced CD34⁺ cells. Isolation of genomic DNA from patient leukocytes and PCR amplification and analysis were performed as described previously.11 To develop quantitative standards, mixtures of genomic DNA extracted from CEM cells containing one proviral copy of either the LN or the L-RRE-neo vectors were diluted in genomic DNA extracted from nontransduced cells. These standards plus multiple samples of DNA from nontransduced PBMC were analyzed with each assay of patient samples. A single pair of PCR primers was made to simultaneously detect the presence of sequences from the LN and L-RRE-neo vectors. The 5′ primer was complimentary to the MoMuLV Psi region (5′-CGAGACCTCATCACCCAGGTTAAG-3′ sense) and the 3′ primer was complimentary to the neo gene (5′-CATCAGAGCAGCCGATTGTCTG-3′ antisense). This pair of primers produces a product of 391 bp from the LN vector (N) and 451 bp from the L-RRE-neo vector (R); the increase in the L-RRE-neo product being caused by the presence of the RRE sequences immediately 5′ of the neo gene, within the span of the primer pair. An oligonucleotide (5′-TCGATCCTCCCTTTATCCAGCC-3′) complimentary to a sequence present in the resultant PCR products from each vector was labeled with -³²P-dATP and used as probe.