Target amplification methods

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. 

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. 

An enhancement of SPR signal intensity was observed by the addition of the antibody to the divalent antigen-antibody complex immobilized onto the surface of the sensor chip, indicating the formation of linear supramolecules. An amplification method of the detection signals for a target molecule has been... 

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]... 

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... 

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... 

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. 

Several diagnostic tests are available to detect acute HCV infection through detection of antibodies or viral target amplification. Antibody detection methods include enzyme immunoassay (EIA) and the recombinant immunoblot assay (RIBA). Specific antibodies to HCV by EIA are positive in only 50% to 70% of patients during the initial onset of symptoms, but 90% of patients have HCV antibodies after 3 months. A number of viral antigens are included in the current version of EIA, resulting in a 99% sensitivity and specificity for detection of HCV antibodies in immunocompetent patients. Patients with autoimmune disorders may have a false-positive EIA and no detectable HCV RNA, in which case the RIBA may be used as a supplemental test to rule out HCV. Viral target amplification techniques are used to detect HCV RNA either qualitatively or quantitatively. Qualitative tests are more sensitive with a detection limit of up to 100 copies/mL and should be reserved to determine spontaneous clearance of acute infection. Spontaneous clearance of HCV can occur in 50% of patients within 3 months of the acute onset of symptoms. ... 

Nucleic acid amplification methods are now considered a standard laboratory tool. They have had a tremendous impact on the diagnosis and treatment of infectious diseases. These highly sensitive methods have the capability to detect and quantitate minute amounts of target nucleic acid in a rapid manner. The polymerase chain reaction (PCR)... 

How an extract was produced from a sample and prepared for hybridization, e. g., extraction method, whether total RNA, mRNA, or genomic DNA was extracted, exogenous sequences (spikes) added (for each spike added type, name/clone id, quantity added), target amplification (RNA polymerases, PCR). 

A third type of contamination is unique to PCR and other amplification methods, such as the ligase chain reaction. It involves the inadvertent contamination of a new reaction with the aerosolized products of a previous reaction. As shown in Table 2, as little as 10"7 pi of a tube of amplified DNA can contain 103 molecules of target (C4). Recommended precautions (K13) involve the use of positive-displacement pipets and the physical separation of areas where PCR reactions are analyzed from those where new reactions are setup. In laboratories that use these precautions, contamination is infrequent, and, when it does occur, is usually at the 1- to 100-molecule level. However, in addition to these procedural measures, it would be useful to have chemical or enzymatic methods of selectively inactivating amplified DNA—similar to the sterilization procedures used to inactivate large numbers of cultured viruses or bacteria. 

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. 

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