Nucleic acid amplification (NAA) methods are now numerous and varied. In general, they can be categorized into two broad categories: target amplification and signal amplification. The chief difference between these two categories is that in target amplification methods, the nucleic acid sequence of interest is geometrically replicated into potentially millions of copies. Signal amplification methods do not increase the number of copies of the target sequence that are present in the sample. Large quantities of signal bind to the target sequence so that the signal becomes measurable. The following table summarizes some of the available techniques. This table is not intended to provide an exhaustive list of all possibilities.
|Polymerase chain reaction (PCR)||Probe hybridization assay (Digene)|
|Ligase chain reaction (LCR)||Branched DNA technologies (b-DNA)|
|Transcription mediated amplification (TMA)||Cleavage based signal amplification (Invader®)|
|Strand displacement amplification (SDA)|
|Reverse transcriptase-polymerase chain reaction (RT-PCR): Amplifies RNA targets|
In addition to NAA, fluorescence in situ hybridization (FISH) techniques, employing nucleic acid probes, represent another useful strategy. FISH assays do not involve amplification and are described in detail in a later section.
Although many alternatives have been developed, PCR and PCR-derived techniques remain the most widely used, primarily because of their simplicity and flexibility. Of the target amplification methods, PCR is the principle for many assays. In some ways, the other methods could be considered "variations on the theme." Although it is beyond the scope of this course to describe in detail each and every technique, the essentials of PCR and RT-PCR will be reviewed.