PubMed · 10476562
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S C Quek, A Singer. 1999. Cervical screening: making it better.. https://pubmed.ncbi.nlm.nih.gov/10476562/
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Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.
Cancer develops through the successive accumulation and selection of genetic and epigenetic alterations, enabling cells to survive, replicate and evade homeostatic control mechanisms such as apoptosis and antiproliferative signals. This transformation process, however, may create vulnerabilities since the accumulation of mutations can expose synthetic lethal gene interactions and oncogene-driven cellular reprogramming ('addiction'), giving rise to new therapeutic avenues. With the completion of the human genome project, it is anticipated that the identification and characterization of genetic networks that regulate cell growth, differentiation, apoptosis and transformation will be fundamental to decoding the complexity of these processes, and ultimately, cancer itself. Genomic methodologies, such as large-scale mRNA profiling using microarrays, have already begun to reveal the molecular basis of cancer heterogeneity and the clinical behavior of tumors. The combination of traditional cell culture techniques with high-throughput screening approaches has given rise to new cellular-genomics methodologies that enable the simultaneous interrogation of thousands of genes in live cells, facilitating true functional profiling of biological processes. Among these, RNA interference (RNAi) has the potential to enable rapid genome-wide loss-of-function (LOF) screens in mammalian systems, which until recently has been the sole domain of lower organisms. Here, we present a broad overview of this maturing technology and explore how, within current technical constraints, large-scale LOF use of RNAi can be exploited to uncover the molecular basis of cancer--from the genetics of synthetic lethality and oncogene-dependent cellular addiction to the acquisition of cancer-associated cellular phenotypes.