Advanced Techniques in Diagnostic Microbiology

212 D. Zhang et al.
TABLE 13.1. Comparison of probe amplification technologies.
Property RCA RAM Q-beta SMART Invader LCR CPT
Amplification 1U 2 u 2 n 2 n 1U 2 n 1U
capability
Temperature − − − − − + −
alteration
Detection of + + + + + + +
DNA target
Detection of + + + + ± + −
RNA target
Detection of + ± − − − ± −
protein
target
Real-time + + + + + ± +
Enzyme used DNA pol DNA pol RNA-RNA pol DNA-RNA pol cleavase ligase RNase H
On-surface + ± − − − − −
amplification
Multiplexing + + ± ± + ± +
SNP detection + + + ± + ± ±
RCA, rolling circle amplification; RAM, ramification amplification; SMART, signal-mediated amplification
of RNA technology; LCR, ligase chain reaction; CPT, cycling probe technology; u, number
of rounds accomplished by DNA polymerase along a C-probe; n, number of cycle; SNP, single nucleotide
polymorphism; DNA pol, DNA polymerase; RNA-RNA pol, RNA directed RNA polymerase;
DNA-RNA pol, DNA directed RNA polymerase.
closed circular molecule is helically twisted around the target strand (Nilsson et al.,
1994). The permanently locked C-probe permits stringent washing for the removal
of unbound components, thereby enhancing assay signal to noise ratios.
The unique design of the C-probe allows its amplification by a rolling circle
(RCA) mechanism as observed in in vivo bacteriophage replication (Fig. 13.1B)
(Fire and Xu, 1995; Baner et al., 1998; Zhang et al., 2001). In this scheme, a single
forward primer complementary to the linker region of the C-probe and a DNA
polymerase bearing strand displacement activity are employed. The polymerase
extends the bound primer along the closed C-probe for many revolutions and
displaces upstream sequences, producing a long single-stranded DNA (ssDNA) of
multiple repeats of the C-probe sequence that can be as long as 0.5 megabase (Baner
et al., 1998). This type of amplification, however, only results in linear growth of
the products with up to several thousand-fold amplification (Baner et al., 1998).
Some of the properties of RCA are summarized in Table 13.1.
Because the product of RCA remains attached to the primer, RCA is amenable to
an on-chip probe amplification system (Fig. 13.2A). In this way, the target molecule
can be recognized, amplified, and detected directly on a solid support, such as a
microarray platform. With RCA, Nallur et al. (2001) were able to detect 480 fmol
(150 molecules) of spotted primers, corresponding to an 8000-fold increase in
detection sensitivity over hybridization under the same conditions. This level of
amplification by RCA on microarray was comparable to that achieved in solution
phase format, indicating that RCA can function with virtually 100% efficacy when