Transcription-based amplification methods

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... 

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). 

Liu QY, Sooknanan RR, Malek LT, Ribecco-Lutkiewicz M, Lei JX, Shen H, Lach B, Walker PR, Martin J, Sikorska M (2006) Novel subtractive transcription-based amplification of mRNA (STAR) method and its application in search of rare and differentially expressed genes in AD... 

In addition to PCR, there are many other technologies to amplify nucleic acids. For example, ligation-based amplification or ligase chain reaction uses sequence-directed oligonucleotide primers and thermostable DNA ligase to assay point mutations, deletions, or insertions in DNA. Strand-displacement amplification uses the inherent strand-displacement activity of DNA polymerases to conduct DNA amplification at a constant temperature. Transcription-based methods such as nucleic acid sequence-based amplification (NASBA) involve in vitro RNA transcription. NASBA and most other transcription-based... 

Reverse transcription (RT)-PCR ISH is based on RT-PCR. The transcripts in a cell are used to make cDNA by reverse transcription, followed by PCR amplification of the cDNA. Details of these methods and representative applications are covered in Chapter 49. 

Recently, PCR-based methods permitting massive amounts of amplification have been shown to allow good preservation of mRNA representation, even from an individual cell s worth of transcript (9,20). In a similar fashion, we successfully... 

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