Target amplification methods polymerase chain reaction

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 number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

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. 

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)... 

In biomedical diagnostics, the amplification of captured or adsorbed nucleic acids using the classical polymerase chain reaction (PCR) method is one of the aims targeted in various biological applications. The enzymatic amplification of desired nucleic acids is often performed after the desorption or release step. Thanks to hydrophilic, highly hydrated magnetic and non-magnetic latex, direct amplification of adsorbed nucleic acid molecules onto the particles [17] is now possible. 

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