10 ml red top for serum 
analysis: see 6.212 
above 
Note: 
For post-transplant samples Southern blot analysis will 
only be performed on samples which demonstrate the neo R 
gene by PCR. 
If samples from relapsed patients contain <75% blasts 
analysis will be performed on specimens after enrichment 
by cell sorting. 
6.3 Study Parameters - Specimen Analysis Protocols . The 
methods for specimen analysis are detailed in 
Appendices 3, 4, 5, 6 and 7. They are summarized 
briefly here. 
Polymerase Chain Reaction The techniques used for 
polymerase chain analysis has been previously 
published by the protocol authors and a reprint is 
available in Appendix 4. In brief, untreated and 
LNL-6 transduced cells are processed in identical 
fashion. 1-2 ug of genomic DNA in a total volume of 
100 ul was used for each reaction. The reaction 
reagents were purchase in kit form (GeneAmp) from 
Perkin Elmer Cetus. The oligonucleotide primers 
used for the analysis of the Neo R gene primer had 
the sequences: CAAGATGGATTGCACGCAGG and 
CCCGCTCAGAAGAACTCGTC and the 4070A envelope region 
have the following sequences: ACCTGGAGAGTCACCAACC 
and TACTTTGGAGAGGTCGTAGC . Samples were subjected to 
30 cycles of denaturation (94°C, 2 min 45 sec), 
annealing (53°C, 2 min), and polymerization (72°C, 
3 min 3 0 sec) in an automatic DNA Thermal Cycler 
then subjected to ethanol precipitation and 
electrophoresed on a 3% NuSieve agarose/1% regular 
agarose gel. Southern blot analysis (using a 32 P end 
labeled oligonucleotide probe) was used to confirm 
the presence of the neo R gene or the 4070A viral 
envelope. These techniques have been used in 
clinical RMGT trials (Appendix 3) . 
Southern Blot Analysis Genomic DNA will be 
prepared and blotted as previously described and 
used in Appendix 5 and 6. Restriction enzymes to be 
used will be Kpnl (cuts within each LTR) and EcoRI 
Recombinant DNA Research, Volume 15 
