Single-cell PCR analysis of the housekeeping genec-raf-1 (A) and the bcl-2/JH rearrangement (B) by seminested PCR analysis of cells isolated from frozen sections of OCI LY8 (lanes O: 1 through 3) and HSB-2 (lanes H: 1 through 3) tumor cells and of small lymphocytes from lymph node involved by HD (lanes L: 1 through 3). The amplified DNA was separated on 2% agarose/NuSieve gel electrophoresis and visualized by staining with ethidium bromide. Ø, molecular weight markers; BL (blank), PCR amplification without DNA template to rule out contamination; C+, positive control (OCI LY8 tumor DNA). C-raf-1 gene has been successfully amplified from 8 of 9 isolated single cells (A) as demonstrated by the presence of a band of 180 bp in 8 of 9 micromanipulated single-cell DNA, which indicates the validity of our methodology for the single-cell PCR amplification. As expected, only OCI LY8 tumor cells showed rearranged bcl-2/JH bands within the expected range of size (110 to 360 bp; lanes O: 1 through 3) (B).
