Real-Time PCR Systems
Some of the limitations of end-point PCR have been addressed in real-time PCR systems, a number of which are now on the market. These systems offer many general technical advantages, including reduced probabilities of variability and contamination, as well as online monitoring and the lack of need for post reaction analyses. Further, some of these systems were developed with contemporary applications such as quantitative PCR, multiplexing, and high-throughput (HT) analysis in mind. In addition, application of real-time PCR has provided significant methodological benefits and improved patient outcomes. In the decade following the first publication on real-time PCR, thousands of papers have appeared in the literature. The range of application is immense and has been partially fulfilled by the availability of lower-cost instruments and reagents. Real-time quantitative PCR is a highly sensitive method that is especially useful for evaluating RNA fingerprints obtained from short interfering RNA (siRNA) experiments and for scientists using RNA interference (RNAi) for mapping cellular pathways.
There are currently five main chemistries used for the detection of PCR product during real-time PCR: DNA binding fluorophores, 5' endonuclease, adjacent linear and hairpin oligoprobes, and the self-fluorescing amplicons. In real-time quantitative PCR techniques, signals (generally fluorescent) are monitored as they are generated and are tracked after they rise above background but before the reaction reaches a plateau. Initial template levels can be calculated by analyzing the shape of the curve or by determining when the signal rises above some threshold value.
Of the important applications is the combination of real-time PCR with either laser capture microdissection or nucleic acids from paraffin-fixed archival samples or whole-transcript amplification from very small numbers of cells. It will be possible to measure gene expression or DNA copy number in specific cell types that are available only in a small quantity. Real-time Q-PCR can be applied to analysis of clinical samples to help stratification of patients in personalized medicine approach. The safety of cell-derived biological compounds or quantification of retrovirus-like particles will be enhanced with real-time Q-PCR. It will also be useful for identification of potential contaminants during the production of recombinant monoclonal antibodies (MAbs) therapeutic use. Combining techniques for sorting fetal cells or DNA from the maternal circulation with Q-PCR will enable early minimally invasive prenatal diagnosis of numerous congenital disorders. Confirmation of expression levels of selected genes from microarray experiments will continue to be conducted using real-time PCR methods. Real-time PCR can be incorporated in the development of highly specific assays that can be performed in the field for use in screening for evidence of biological weapons.
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