Degenerate oligonucleotide-primed PCR (DOP-PCR): evaluation of its reliability for screening of genetic alterations in neoplasia.

Genetic alterations underlie the development and progression of neoplasia. Screening for genetic alterations in biopsies and premalignant lesions, where the neoplastic tissue analyzed usually constitutes only a small fraction of the cells in the specimen, often is difficult and tedious. Even after successful isolation of DNA, limited quantities allow only a few genetic analyses, limiting the identification of genetic changes in small lesions and the establishment of molecular cancer progression models. One way to overcome the low DNA abundance problem is to use universal or whole genomic amplification (10). One of the most promising approaches reported for universal genomic amplification is the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) assay (2). This technique allows the amplification of small genomic DNA samples producing severalfold more DNA for genetic analysis (8). DOP-PCR has been successfully used in cytogenetic analyses, including chromosome painting (6) and comparative genomic hybridization (CGH) (9). However, few data have been reported on the use of DOP-PCR in loss of heterozygosity (LOH) and mutation detection. The purpose of our work was to evaluate the reliability of the DOP-PCR method to identify LOH and gene mutations in primary tumor samples. Normal lymphocyte and corresponding tumor DNA from two lung tumors displaying LOH for at least one of the microsatellite markers studied were used for DOP-PCR genomic amplification and subsequent microsatellite analysis. Normal lymphocyte and corresponding tumor DNA from one bladder cancer patient (with a mutation in the PTEN tumor-suppressor gene) and two head and neck tumors (normal and mutated, respectively, for exon 8 of p53) were subjected to DOP-PCR amplification and subsequent PCR and sequencing analysis. Genomic DNA was prepared using standard procedures and quantified spectrophotometrically (7). Serial dilutions of DNA were prepared and used as a template for universal genome amplification. DOP-PCR amplification was performed as previously described (2) using 2 μM of DOP primer (5′-CCGACTGAGNNNNNNATGTGG -3′), 200 μM dNTPs, 0.1% Triton X100, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2 and AmpliTaq DNA Polymerase (Perkin-Elmer, Norwalk, CT, USA) under mineral oil in a