Key Issues, Challenges and Future Opportunities in Gene Quantification.- I: Methods/Technology Issues.- A. Gene Quantitation Based on PCR Amplification.- Present and Future Detection Formats for PCR Quantitation of Nucleic Acids.- Determination of Target Copy Number of Quantitative Standards Used in PCR Based Diagnostic Assays.- Quantification of Specific Nucleic Acids, Regulated RNA Processing and Genomic Polymorphisms Using Reversed-Phase HPLC.- Capillary Electrophoresis for Quantitative Genetic Analysis.- Quantitative PCR Technology.- Statistical Estimations of PCR Amplification Rates.- Fluorescence Monitoring of Rapid Cycle PCR for Quantification.- Kinetic Elisa-PCR: A Versatile Quantitative PCR Method.- B. Gene Quantitation Based on Other Target Amplification Systems.- Quantitation of RNA by NASBA(TM): Applications and Issues for HIV-1 and AIDS.- Application of Transcription-Mediated Amplification to Quantification of Gene Sequences.- C. Gene Quantitation Based on Signal Amplification.- Branched DNA (bDNA) Technology for Direct Quantification of Nucleic Acids: Design and Performance.- Hybrid Capture(TM) - A Sensitive Signal Amplified Test for the Detection and Quantitation of Human Viral and Bacterial Pathogens.- II: Applications.- Quantification of Gene Expression by Competitive RT-PCR: The hCG?/LH? Gene Cluster.- Quantitative Detection of Mycoplasma DNA Using Competitive PCR.- The Detection and Quantification of bcr-abl in Chronic Myeloid Leukemia Following Marrow Transplantation.- Competitive RT-PCR Analysis of Brain Gene Expression During Inflammation and Disease.- Development and Application of Real-Time Quantitative PCR.- Branched DNA (bDNA) Technology for Direct Quantification of Nucleic Acids: Research and Clinical Applications.- Quantification of Plasmid DNA Expression in Vivo.
Geneticists and molecular biologists have been interested in quantifying genes and their products for many years and for various reasons (Bishop, 1974). Early molecular methods were based on molecular hybridization, and were devised shortly after Marmur and Doty (1961) first showed that denaturation of the double helix could be reversed - that the process of molecular reassociation was exquisitely sequence dependent. Gillespie and Spiegelman (1965) developed a way of using the method to titrate the number of copies of a probe within a target sequence in which the target sequence was fixed to a membrane support prior to hybridization with the probe - typically a RNA. Thus, this was a precursor to many of the methods still in use, and indeed under development, today. Early examples of the application of these methods included the measurement of the copy numbers in gene families such as the ribosomal genes and the immunoglo bulin family. Amplification of genes in tumors and in response to drug treatment was discovered by this method. In the same period, methods were invented for estimating gene num bers based on the kinetics of the reassociation process - the so-called Cot analysis. This method, which exploits the dependence of the rate of reassociation on the concentration of the two strands, revealed the presence of repeated sequences in the DNA of higher eukaryotes (Britten and Kohne, 1968). An adaptation to RNA, Rot analysis (Melli and Bishop, 1969), was used to measure the abundance of RNAs in a mixed population.
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