Fig. 1.

Genotype analysis in the three different tumor cell lines. DNA was generated from SCCHN cell lines. a Three different tumor cell lines were cultured and harvested as described in “Materials and methods” for PCR amplification with the primers 5′-ATC TCA AGT CCC CCT TGC CG-3′ and 5′-GCA ACT GAC CGT GCA AGT CA-3′. An initial denaturation step was used at 95°C for 5 min, 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. PCR product (296 bp fragment) was digested with BstUI (New England Biolabs, Inc., Beverly, MA, USA) overnight at 60°C, which revealed the presence two bands of an arginine (visible fragment 169 and 127 bp)-encoding allele (BstU1 sensitive, [18]) or a proline-encoding allele (BstU1 insensitive), as visualized on ethidium bromide-stained agarose gel electrophoresis. b These three SCCHN cell lines (SCC-4 not shown) were sent for direct fluorogenic sequencing analysis of both coding and non-coding strands. Subsequent BLAST alignment was used to determine the genotype of the sequence of codon 72 of the TP53 exon 4, and the homozygosity of PCI-13 (R/R) and heterozygosity of SCC-90 (R/P) were confirmed