Isothermal target amplification

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... 

Zhang DY, Zhang W, Li X, Konomi Y. Detection of rare DNA targets by isothermal ramification amplification. Gene 2001 274 209-16. 

In vitro transcription is an important technique for the preparation of RNA that can be used for various purposes. At present there are no efficient simple techniques available for the synthesis of RNA oligonucleotides comparable to those of current automated DNA synthesis. In vitro transcription is also the basis for efficient isothermal gene amplification techniques. In vitro transcription can be performed in one of the two formats (Fig. 7.5) by the vector-based approach in which a cloned target (template) DNA is abutted to the T7 promoter in a linearized plasmid (or phagemid) or by the synthetic oligonucleotide-based approach in which the template DNA is linked to a mobile promoter. 

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

Wave [8], Approximately 50 to 150 ng of DNA is required. Following amplification, two simultaneous Invader reactions (isothermal) are performed per variant. Specifically, a three-stranded structure is formed through hybridization of two Invader probes to complimentary regions of the target DNA. The three-stranded structure is comprised of the target DNA and two probes, one of which overlaps by one base with the other two, thus creating a flap on the 5 end that does not hybridize. A proprietary cleavase... 

We have exemplified BART applications in molecular IVDs and demonstrated its compatibility with various amplification methods, its ability to detect and quantify different targets, to cope with crude sample preparations and to provide rapid results under challenging conditions. Overall, BART is a universal reporter system for any molecular in vitro diagnostic tests based on isothermal nucleic acid amplification techniques. 

NASBA technology depends on selective primer-template recognition to drive a cyclic, exponential amplification of the target sequence (1). Notably, NASBA operates continuously under isothermal conditions and consequently achieves rapid amplification using only standard laboratory equipment. 

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