Breakpoint identification by PAMP with an INK4A minigenomic tiling array.

(A) Five groups of primers (F_A, F_B, R_x, R_Y and R_Z, the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3]. The mapped CDKN2A breakpoints of the Detroit 562 cell line (Figure 5) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F_A, F_B, R_Y and R_Z) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2.