Advanced Techniques in Diagnostic Microbiology

12. Non-PCR Amplification 193
and contaminated matrices including formalin-fixed tissues (Wang et al., 2004b).
The basic method involves restriction endonuclease digestion of total DNA and circularization
of fragments with DNA ligase. After elimination of noncircularized
DNA by exonuclease digestion, the mixture is amplified using random primers
and Phi 29 Polymerase (Dean et al., 2001). Examination of the products showed a
balanced genome coverage exceeding that of balanced PCR amplification (Wang
et al., 2004a, 2004b).
An even greater lever of amplification of circular DNA probes can be achieved
using circle-to-circle amplification (C2CA), which provides a means for more than
a10 8 -fold amplification (Dahl et al., 2004). DNA circles are first generated in a
basic RCA reaction using padlock probes (Nilsson et al., 1994) (Fig. 12.2E) to
generate single-stranded concatenated products (Fig. 12.3A). After heat inactivation
of the polymerase, an excess of the complementary replication primer is
hybridized and the concatenated products can be monomerized using an restriction
enzyme that cuts in the dsDNA region formed by the binding of the complementary
primer to the linear strand (Fig. 12.3B). The restriction enzyme is then heat
inactivated, which allows both primers to dissociate and re-anneal at each end of
the linear template. DNA ligase is then added to reform the circular template that
can be used for additional rounds of replication (Fig. 12.3C) (Dahl et al., 2004).
Each round of a C2CA reaction is a linear amplification cycle so the reaction
can be precisely quantified. Because the circles are of a defined polarity, this can
facilitate hybridization-based downstream processing or quantified in real-time
(Lizardi et al., 1998). Dahl et al. also demonstrated multiplexed genotyping using
the C2CA reaction (Dahl et al., 2004). This system is robust in its design, and the
accuracy and fidelity of replication is maximized using the Phi 29 DNA Polymerase
(Blanco et al., 1989).
Anchored and Ligation-Mediated RCA Technologies
The use of immobilized nucleic acids and proteins is now commonplace in both
basic research and diagnostics. The general methodology used for signal detection
involves the passive hybridization of a detector probe and can limit the sensitivity
of the assay depending on target abundance. Accordingly, RCA has been
successfully applied to immobilize oligonucleotide targets to increase sensitivity
and signal intensity. Immobilized RCA also takes advantage of the nondiffusible
nature of the RCA concatameric product. RCA assays have been developed for
SNP and mutation analysis (Christian et al., 2001; Pickering et al., 2002; Alsmadi
et al., 2003), and the amplification mechanism is useful for protein microarrays
(Schweitzer et al., 2000, 2002; Zhou et al., 2004).
The basic methodology of immobilized RCA was initially developed using a
biotinylated oligonucleotide primer that could anneal to the ends of a circular probe.
This formed a double-stranded complex that could be attached to streptavidin beads
(Hatch et al., 1999). If the hybridization of the ends was perfect, this would allow
DNA ligase to covalently join the two ends; therefore; polymerization from an
external circle primer would occur. If the ends were not perfectly complementary,