An integrated physical and genetic map of the nematode Pristionchus pacificus.

The free-living nematode Pristionchus pacificus is one of several species that have recently been developed as a satellite system for comparative functional studies in evolutionary developmental biology. Comparisons of developmental processes between P. pacificus and the well established model organism Caenorhabditis elegans at the cellular and genetic levels provide detailed insight into the molecular changes that shape evolutionary transitions. To facilitate genetic analysis and cloning of mutations in P. pacificus, we previously generated a BAC-based genetic linkage map for this organism. Here, we describe the construction of a physical map of the P. pacificus genome based on AFLP fingerprint analysis of 7747 BAC clones. Most of the SSCP markers used to generate the genetic linkage map were derived from BAC ends, so that the physical genome map and the genetic map can be integrated. The contigs that make up the physical map are evenly distributed over the genetic linkage map and no clustering is observed, indicating that the physical map provides a valid representation of the P. pacificus genome. The integrated genome map thus provides a framework for positional cloning and the study of genome evolution in nematodes.

Comparison of the RNA-amplification based methods RT-PCR and NASBA for the detection of circulating tumour cells.

Increasingly, reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect clinically significant tumour cells in blood or bone marrow. This may result in a redefinition of disease-free and clinical relapse. However, its clinical utility may be limited by lack of automation or reproducibility. Recent studies have suggested nucleic acid sequence-based amplification of target RNA may be more robust. In this study, nucleic acid sequence-based amplification was established to detect melanoma, colorectal and prostate cancer cells. Nucleic acid sequence-based amplification and RT-PCR both successfully amplified target RNA in peripheral blood samples from patients with melanoma and colorectal cancer, but only RT-PCR detected PSA in blood samples from patients with prostate cancer. There was relatively good agreement between sample replicates analyzed by RT-PCR (Kappa values of one for tyrosinase, 0.67 for CK-20 and one for PSA), but less agreement when analyzed by nucleic acid sequence-based amplification. This may limit the routine use of NASBA for the detection of clinically significant disease. In summary, RT-PCR appears at present to be the most reliable and reproducible method for the detection of low-level disease in cancer patients, although prospective studies are warranted to assess the clinical utility of different molecular diagnostic methods.

Comparison of immunocytochemistry, reverse transcriptase polymerase chain reaction, and nucleic acid sequence-based amplification for the detection of circulating breast cancer cells.

Detection of tumor cells in blood and bone marrow is increasingly used for the staging of patients with breast cancer and to evaluate the presence of tumor cells in peripheral blood progenitor cell collections to be used after high-dose therapy. We evaluated the sensitivity and specificity of three different methods for detection of tumor cells among non-tumor tissue. An immunocytochemical assay using antibodies directed against epitopes of the cytokeratin-19 (CK19) protein and two RNA-based methods: reverse transcriptase polymerase chain reaction (RT-PCR) and Nucleic Acid Sequence-Based Amplification (NASBA) for the same target gene were tested. With all the three methods, false-positive results were observed when peripheral blood mononuclear cells (PBMC) of healthy volunteers were tested. There was no concordance between the RNA-based assays and the immunocytochemical assay. The false-positive results in the RNA-based assays may be due to 'illegitimate expression' of epithelial genes in normal PBMC. The false-positive results in the immunocytochemical assay resulted from background staining of monocytes and granulocytes. This study demonstrates that CK19 is not a suitable target to detect the presence of breast tumor tells in PBMC. To reliably detect circulating tumor cells with RNA methods, the selection of suitable target genes is required, which are highly expressed in tumors but not at all in normal cells of blood and bone marrow. Genes with such characteristics may be identifiable with novel differential display techniques.