Transcription-based amplification system

Fahey, E., Kwoh, D. Y. and Gingeras, T. R. Self-sustained sequence replication (3SR) an isothermal transcription-based amplification system alternative to PCR , PCR Methods Appl., 1, 25-33 (1991). 

An amplification reaction that is used to amplify target RNA or denatured DNA is called the transcription-based amplification system (TAS). This technique involves using an enzyme called reverse transcriptase and a primer with sequence complementary to the sample target RNA molecule in order to synthesize a complementary DNA (cDNA) copy of the sample target RNA. After denaturation to separate the strands, another primer and additional reverse transcriptase are added to synthesize a double-stranded cDNA molecule. Since the first primer has also an RNA polymerase binding site, it can, in the presence of T7 RNA polymerase, amplify the double-stranded cDNA to produce 10 to 100 copies of RNA. The cycle of denaturation, synthesis of cDNA, and amplification to produce multiple RNA copies is repeated. With as few as four cycles, a 2- to 5-millionfold amplification of the original sample RNA target is possible. However, the time required to achieve a millionfold amplification is approximately 4 hours, which is the same amount of time required by PCR. The TAS requires, however, the addition of two enzymes at each cycle and, as such, can be cumbersome. 

Transcription-based amplification system (TAS) Target. . Reverse transcriptase RNA polymerase Yes... 

Kwoh DY, Davis GR, Whitfield KM, ChappeUe HL> DiMichele LJ, Gingeras TR. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad SciUSA 1989 86 1173-7. 

A protein microarray relies on high-throughput amplification of each predicted ORF by using gene-specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system. The protein products from the unpurified reactions are printed directly onto nitrocellulose microarrays without further purification [113]. 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

Zhou et al. [175] described the determination of severe acute respiratory syndrome (SARS) coronavirus by a microfluidic chip system. The unit included an LIF microfluidic chip analyzer, a glass microchip for both PCR and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. According to the authors, the system allowed efficient DNA amplification of the SARS coronavirus followed by electrophoretic sizing and detection on the same chip. 

Kimoto M, Yamashige R, Yokoyama S, Hirao I (2012) PCR amplification and transcription for site-specific labeling of large RNA molecules by a two-rmnatirral-base-pair system. J Nucleic Acids 2012 230943. doi 10.1155/2012/230943... 

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