Table 4.9-B. Detection of Ela sequences in the respiratory epithelium of 
individuals with cystic fibrosis. Respiratory epithelial cells were ob- 
tained from normals and individuals with cystic fibrosis (CF) from the 
inferior turbinates of the nose (by direct visualization) and bronchial 
epithelial cells from trachea and/or main bronchi by fiberoptic bronchosco- 
py, using a standard cytology brush. Neutrophils, lymphocytes and monocytes 
were isolated from the blood. Cells were immediately suspended in RPMI 
1640, collected by centrifugation and lysed in 4 M guanidinium thiocyanate, 
25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M /3-mercaptoethanol . Cell 
number was determined in the cell lysate by quantifying the number of Alu 
sequences present in the DNA in an aliquot of the cell lysate compared with 
a standard curve of highly purified human genomic DNA (assuming 7.2 pg of 
DNA per cell). To detect Ela sequences, the DNA in an aliquot of cell 
lysate was amplified by the polymerase chain reaction (PCR) using Taq DNA 
polymerase and Ela specific primers (Adl7 : 5 ' -GAGACATATTATCTGCCACGGAGG- 3 ' 
and Adl8 : 5 ' -TTGGCATAGAAACCGGACCCAAGG- 3 ' ; the sequences of both primers are 
100% homologous to type 2 and type 5 adenoviruses) for 35 cycles (94° -1 
min, 65° -1 min, 72° -1 min) (Gingeras et al., 1982; Van Ormondt et al . , 
1978). In parallel with each PCR amplification, a standard curve was con- 
structed using increasing amounts of adenovirus type 2 DNA. An aliquot of 
PCR product of each sample was bound to slot-blot nylon filter and hybrid- 
ized with a 32 P- labeled "nested" Ela probe. A total of 91 normal subjects 
and 43 individuals with CF were studied. 
Recombinant DNA Research, Volume 16 
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