Target amplification

Many nucleic acid detection strategies use target amplification, signal amplification or both. Invader, branched DNA (bDNA) and rolling circle amplification (RCA) are three approaches. 

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... 

PCR and related target amplification systems typically employ a single pair of primers. Each primer is usually 20 to 40 bases in length and anneals to the complementary sequence on the target nucleic acid to initiate the amplification reac-... 

In summary, bDNA has a number of distinct theoretical and practical advantages over target amplification systems for direct quantitation of specific nucleic acid sequences. The following sections review the specific clinical and research applications of this technology. 

Since rolling circle amplification takes place at a constant temperature, there is no need for the target amplification process to take place in a thermal cycler, which is required to regulate the temperature for different parts of the reaction. The type of DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the PCR-based diagnostics. On the other hand, the RCA method requires the environment to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]... 

It is a truism that accurate pipettes, proper air circulation, and pure chemicals and active enzymes assure success in any analytic procedure. Molecular diagnosis is no exception to this. In addition, because molecular diagnosis often requires exponential target amplification before detection of a sequence of interest, sequestration of amplified materials (post-PCR) from incoming patient samples and preparation... 

Conventionally, the variants are characterized by coamplification with wild-type sequences using reverse transcription polymerase chain reaction (RT-PCR). However, this approach focuses on small regions of the known wild-type mRNA. Because of this threshold detection, spliced transcripts expressed at low levels may fall below the threshold of detection. To avoid this and other limitations of the conventional RT-PCR technique, the targeted amplification method can be used (Poola et al., 2000). This method involves the targeted amplification of the alternatively spliced molecules as separate gene populations using specific primers designed for the alternative splice junctions, without coamplification of wild-type molecules. 

A better method for highly parallel genetic analysis is needed. One with single molecule sensitivity that eliminates both the requirement for target amplification and the need for a target to be chemically modified or labeled for its detection would be ideal. Innovations in array construction, sample nucleic acid preparation... 

Hertzberg M, Sievertzon M, Aspeborg H, Nilsson P, Sandberg G, Lundeberg J. cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol. Plant J. 2001 25 585-591. 

When the amount of target nucleic acid is increased by synthetic in vitro methods, target amphfication is said to occur. The polymerase chain reaction (PCR) is the best known and most widely applied of the target amplification methods. Because of the commercial availability of thermostable DNA polymerases, kits, and instrumentation, this method has been widely adopted in research and is also rou-tmely used in the clmical laboratory. 

A large number of other methods of target amplification have been described. Short descriptions of some of these methods follow, with citations of sources of further information. 

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... 

It is not always necessary to amplify the target DNA sequence or a cDNA sequence complementary to an RNA target. Instead of target amplification, both signal amplification and probe amplification can be used. 

Q-beta replicase is a method in which the concentration of probe increases if the target is present. Similar to target amplification, a large amount of nucleic acid product makes detection much easier. An RNA probe is replicated exponentially in the presence of the RNA-directed RNA polymerase, Q-beta replicase. The probe is a recombinant RNA hybrid that includes sequence complementary to the target embedded in a naturally occurring template for the enzyme. 

---