The Basic Concept

Retroviral replication relies on the use of reverse transcriptase to make a complementary DNA (cDNA) copy from its RNA target(s) (Varmus, 1988). Reverse transcriptase has two enzymatic activities that are important for cDNA formation; that is, a DNA polymerase activity and an RNAse H activity. The retroviral cDNA synthesis reaction relies on the presence of a virion-packaged cellular tRNA molecule that serves as a primer for first-strand DNA synthesis. The RNAse H activity degrades the initial RNA template so the first-strand cDNA can then be used as a template for an additional round of reverse transcription to make the second-strand DNA. The dsDNA "genome" now serves as a transcriptional template for the synthesis of viral proteins as well as new genome copies that will be packaged in viral particles (Varmus 1988). Adaptation of this reaction sequence to the laboratory was attractive because it could be used to target RNA directly (Guatelli et al., 1990; Compton, 1991). Target amplification would rely on primers that substitute for the retroviral tRNA molecules (Guatelli et al., 1990; Compton 1991).

The original publication describing the transcription-based amplification reaction used AMV RT (avian myeloblastosis virus reverse transcriptase), bacteriophage T7 RNA polymerase, and E. coli RNAse H (Guatelli et al., 1990). The first step or "noncyclic" phase of the reaction begins with first-strand cDNA synthesis initiated from a primer (P1) containing a T7 RNA polymerase promoter sequence at its 5' end and a target-specific sequence at the 3' end (Fig. 12.1). Reverse transcriptase extends the primer to yield a first-strand "anti-sense" cDNA contained within an RNA:DNA hybrid. RNAse H hydrolysis of the RNA template enables the second-primer (P2) to anneal and prime second-strand cDNA synthesis using the first cDNA strand as a template. This results in the production of a dsDNA template for the DNA-dependent T7 RNA polymerase (Fig. 12.1). Transcription from a dsDNA template containing the T7 promoter can produce 10-1000 copies of antisense RNA (Dunn and Studier, 1983), and they themselves serve as templates for additional rounds of replication. Primer P2 can also incorporate a T7 promoter sequence to enable the generation of transcripts from both ends of the dsDNA molecule (Compton, 1991). The T7 promoter sequences may be substituted by others such as SP6 (Brown et al., 1986) and T3 (Bailey et al., 1983).

The self-sustaining nature of the reaction occurs through successive cycles as primers P1 and P2 are used for first-strand cDNA synthesis from anti-sense and sense transcripts, respectively. The resultant cDNA copies are then primed for

  • d Isothermal System
  • d Isothermal System

Figure 12.1. A standard transcription-based isothermal system. The basic principle of this technique is the introduction of a bacteriophage RNA polymerase promoter to end of the cDNA generated by reverse transcription. The first-strand cDNA is reverse-transcribed from primer P1 and then replicated through the DNA polymerase activity of RT using a second primer (P2). This results in a dsDNA molecule that is a substrate for RNA poly-merase. This sequence of events generates a self-sustained reaction consisting of simultaneous rounds of transcription, reverse transcription, and DNA polymerization to yield an exponential amplification of the target RNA within 10-15 min. This assay has been given numerous designations including self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and transcription-based amplification system (TAS). See text for details.

Figure 12.1. A standard transcription-based isothermal system. The basic principle of this technique is the introduction of a bacteriophage RNA polymerase promoter to end of the cDNA generated by reverse transcription. The first-strand cDNA is reverse-transcribed from primer P1 and then replicated through the DNA polymerase activity of RT using a second primer (P2). This results in a dsDNA molecule that is a substrate for RNA poly-merase. This sequence of events generates a self-sustained reaction consisting of simultaneous rounds of transcription, reverse transcription, and DNA polymerization to yield an exponential amplification of the target RNA within 10-15 min. This assay has been given numerous designations including self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and transcription-based amplification system (TAS). See text for details.

dsDNA synthesis using the complementary primer resulting in another template for T7 RNA polymerase transcription (Fig. 12.1). Product accumulation beginning at this stage is exponential and cycles of transcription and cDNA synthesis enable the reaction to enter the "cyclic" phase of amplification (Guatelli et al., 1990). The rapid kinetics of the 3SR reaction can yield a 106-fold amplification in 10 min, whereas a PCR reaction to reach similar magnitude would require about 20 cycles (Guatelli et al., 1990). Background DNA does not interfere with the reaction because single-stranded RNA sequences are specifically targeted. Additionally, because the reaction is carried out at a relatively low temperature, near 40°C (Compton, 1991), this makes it attractive for in situ assays where cell and tissue integrity are important (Mueller, 1997). The latter is a concern especially when the 3SR technique is combined with histochemical staining procedures.

The ability of the 3SR reaction to specifically amplify ssRNA (single-stranded RNA) makes the assay particularly attractive for the detection of viral genomes, mRNA, and rRNA. This extends the range of nucleic acid amplification methods for both diagnostics and research. However, the reaction conditions are isothermal, and this offers unique challenges in terms of probe design and target choice due largely to the potential for RNA to form stable secondary structures. For the development of high-throughput assays, development of real-time amplification methods such as molecular beacons has been particularly challenging (Leone et al., 1998; Szemes and Schoen, 2003).

Fahy and co-workers experimented to optimize standard reaction conditions of substrate concentrations, temperature, pH, and ionic strength (Fahy et al., 1991, 1994). In such a complex reaction mixture, the optimal rNTP and dNTP concentrations of 4 and 0.05 mM, respectively, were much higher than Km values reported for single enzyme reactions (Fahy et al., 1991,1994; Cline et al., 1996). However, the standard reaction conditions of 0.1 |M each primer, 20 mM KCl, 30 mM MgCl2, and 40 mM Tris pH 8 were unremarkable. Interestingly, the RNAse H activity of AMV RT could be enhanced by the addition of 15% DMSO and 15% sorbitol or 10% glycerol to the reaction, and this allowed E. coli RNAse H to be omitted (Fahy et al., 1991). With the further omission of chloride, the temperature of the reaction could be increased from 42°C to 50°C (Fahy et al., 1994). This two-component method was further modified with the substitution of HIV reverse transcriptase. In this case, RNAse H activity was considerably slower, but this led to more homogeneous reaction products and a higher RNA to DNA ratio (Gebinoga, 1996). Significant efforts at the design of primers have been documented including the optimization of the T7 sequence in the context of the reaction, as well as guidelines for the choice of length and composition of the target-specific sequences (Fahy et al., 1991). A general guide to probe design is given in Deiman et al., (2002).

An important consideration in all nucleic acid amplification procedures is to ensure that replication fidelity is maintained, and this criterion is met by the 3SR reaction. Transcription-based systems were demonstrated to give an error frequency of less than 0.3% in cloned DNA products from two different segments of theHIV-1 gag gene (Sooknanan et al., 1994). An overall error rate of 2 x 10-4 was calculated for the combined effects of both polymerases (Sooknanan et al., 1994).

This approximates the error rate of thermostable DNA polymerases, which range from approximately 0.7 x 10-4 for Taq polymerase to 1.6 x 10-6 for PFU and other "proof-reading" polymerases (Tindall and Kunkel, 1988; Brail et al., 1993; Cline et al., 1996). This is especially important for the use of 3SR systems in SNP or mutation detection procedures (Berard et al., 2004).

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