Advanced Techniques in Diagnostic Microbiology

13. Recent Advances in Probe Amplification 211
technology for a particular application, one has to understand the principle of the
technology and address the need of the clinical problem accordingly.
This chapter will review the most common probe amplification technologies and
present some of their applications with primary focus on microorganism diagnosis
in clinical laboratory. For more in-depth discussion of clinical applications, the
readers should refer to other excellent chapters in this book.
Rolling Circle Amplification
Circularizable probe (C-probe or padlock probe) is a uniquely designed oligonucleotide
probe that contains three regions: two target complementary sequences
located at the 5 ′ and 3 ′ termini and an interposed generic linker region (Nilsson
et al., 1994; Zhang et al., 1998). Once the C-probe hybridizes to its target, the 5 ′ and
3 ′ ends are juxtaposed (Fig. 13.1A). A closed circular molecule is then generated
after incubation of the C-probe-target complex with a DNA ligase. The resulting
A: C-probe
B: RCA
C: RAM
FIGURE 13.1. Schematic representation of C-probe, RCA, and RAM. (A) A C-probe hybridizes
to its target through its complementary regions and helical turns formed between
C-probe and target results in the locking of C-probe onto the target. The sequence between
the target-binding regions is generic for the binding of primers. (B) A DNA polymerase
( ) extends a bound primer along a closed C-probe for 5 rounds through rolling circle
amplification (RCA). (C) A forward primer ( ) bound to a C-probe is extended by
DNA polymerase ( ), generating a long ssDNA. Multiple reverse primers ( ) bind to
the nascent ssDNA as their binding sites become available. Each bound reverse primer extends
and displaces the upstream primers and their extended products. The forward primer
binding sites of the displaced ssDNA are then available for the forward primers to bind and
extend similarly, thus forming a large ramifying DNA complex (RAM).