Advanced Techniques in Diagnostic Microbiology

186 M.L. Pendrak and S.S. Yan
TMA (transcription-mediated amplification), TAS (transcription-based amplification
system) (Kwoh et al., 1989), and 3SR (self-sustained sequence replication)
(Fahy et al., 1991, Guilfoyle et al., 1997). All these methods use the same basic
concept for exponential amplification of a target nucleic acid sequence and are
modeled on the strategy of retroviral replication. For purposes of simplifying the
terminology, we will refer to the general method as a 3SR reaction in the following
text.
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 secondstrand
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