Signal versus Target Amplification Systems

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 

The total amplification achieved by PCR is described by the expression, (1 + ) , where E is the average per-cycle efficiency and n is the total number of cycles. The amount of target sequence and the variable presence of inhibitors in clinical specimens influence both the efficiency and the kinetics of amplification. As seen in the preceding expression, small differences in the efficiency of amplification are exponentially compounded and lead to very large and unpredictable differences in product yield. The situation is even more complicated when the target is RNA. PCR must be preceded by reverse transcription to produce complementary DNA (cDNA), and the efficiency of this process is another variable that may influence product yield. 

Competitive PCR (cPCR) has emerged as the best strategy for controlling the sam-ple-to-sample variability of PCR. In cPCR different templates of similar lengths and with the same primer binding sequences are coamplified in the same tube. This ensures identical thermodynamics and amplification efficiency for both templates. The amount of one of the templates must be known and, after amplification, products of both templates must be distinguishable and separately quantifiable. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ ) /Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

In bDNA the number of target molecules is not altered. The signal of direct hybridization rather than the nucleic acid sequence itself is amplified and thus is directly proportional to the amount of target sequence present in the clinical sample. Both RNA and DNA sequences can be measured directly in clinical specimens, and there is no need to transcribe RNA into cDNA as there is with PCR. 

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