Transcription-mediated amplification

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... 

Ender A et al. (2004) Screening of blood donations for HIV-1 and HCV RNA by transcription-mediated amplification assay one year experience. Transfus Med Hemother. 31 10-15. Kasermann F, Kempf C (1997) Photodynamic inactivation of enveloped viruses by buckminster-fullerene. Antiviral Res. 34 65-70. 

Transcription-based amplification methods are modeled after the replication of retroviruses. These methods are known by various names including nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA)/ and self-sustained sequence repfi-cation (3SR) assays. Isothermal target amplification, using the collective activities of reverse transcriptase, RNase H, and RNA polymerase, is common to these methods. As illustrated in Figure 37-6, the method may be applied to... 

C trachomatis and CT, Chlamydia trachomatis N, gomrrhoeae and GC, Neisseria gonorrhoeae PCR, polymerase chain reaction SDA, strand displacement assay TMA, transcription-mediated amplification. 

KT-PCR, Reverse transcription-polymerase chain reaction TMA, transcription mediated amplification bDNA, branched DNA A5R, anaiyte-specific reagent. 

Pasternack R, Vuorinen P, Miettinen A. Evaluation of the Gen-Probe Chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women. J Clin Microbiol 1997 35 676-8,... 

Mahony J, Chong S, Jang D, Luinstra K, Faught M, Dalby D, et al. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated ampUfica-... 

Alcami J, de Lera T, Folgueira L, Pedraza MA, Jacque JM, Bachelerie F, Noriega AR, Hay RT, Harrich D, Gaynor RB et al. Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J 1995 14(7) 1552-1560. 

No conclusive information is as yet available on the mechanism of the initial step of rDNA amplification. It is not known whether the first amplified copy of rDNA is a replica or an excision product. A process whereby amplification is mediated by RNA followed by reverse transcription has also been suggested (Tocchini-Valentini and Crippa, 1971 see later). Furthermore, it remains unknown whether one, a few, or all chromosomal rRNA genes are involved in gene amplification. Two different mechanisms—not necessarily mutually exclusive— have been proposed for the production of additional rDNA replicas (Miller, 1966). The amplified rDNA might be synthesized only on the chromosomal template. Alternatively, the newly synthesized rDNA copies could serve as new templates for subsequent replications, resulting in a cascade effect. Autoradiographic evidence has been presented which favors the second mechanism (Macgregor, 1968). 

The split-ubiquitin system enables signal amplification from a transcription factor-mediated reporter readout (36,37). In one application, the interaction of two membrane proteins forces reconstitution of two halves of ubiquitin, leading to a cleavage event mediated by ubiquitin-specific proteases that release an artificial transcription factor to activate a reporter gene. As discussed, indirect readout of the reporter limits kinetic analysis, and the released transcription factor must translocate to the nucleus. 

---