Advanced Techniques in Diagnostic Microbiology

192 M.L. Pendrak and S.S. Yan
amplification (Fire and Xu, 1995; Lizardi et al., 1998). The simplicity of RCA
has enabled the development of new technologies in target detection and includes
enhanced sensitivity in DNA quantification (Nallur et al., 2001), DNA mutation
detection (Lizardi et al., 1998; Ladner et al., 2001), SNP detection (Qi, et al., 2001;
Pickering et al., 2002), and array-based sandwich immunoassays (Schweitzer et al.,
2000; Schweitzer et al., 2002). Major advancements in SNP and mutation detection,
whole genome amplification and analysis, and amplification using immobilized
oligonucleotides have made RCA one of the most versatile new technologies.
Applications of RCA Techniques
Detection of Single Nucleotide Changes
Identifying genomic mutations and polymorphisms has both current and future
implications in disease detection and prevention. RCA is emerging as a fundamental
technology due to its ability to be adapted to real-time, high-throughput,
and immobilized probe platforms. The main adaptation of RCA in this area is
illustrated in Fig. 12.2E. If a linear ssDNA molecule is added to a denatured template,
the ends of the molecule will be brought together at the target. If properly
hybridized, the two ends can be joined by ligation thus creating an entrance primer
for DNA polymerase replication of the incoming molecule. This has been termed
a padlock probe (Nilsson et al., 1994; Baner et al., 2001; Nilsson et al., 2002).
Using a padlock probe, DNA ligase can accurately discriminate between matched
and mismatched substrates in this region such as for allelic discrimination, SNP
detection, or mutation detection (Luo et al., 1996; Landegren et al., 1988; Faruqi
et al., 2001). By introducing mismatches at the hybridization site, it allows the
system to discriminate between single nucleotide changes between samples for
accurate genotyping (Pickering et al., 2002).
RCA has also been applied to whole genome amplification from small numbers
of cells and is especially useful when dealing with precious clinical specimens.
For instance, ramification amplification (Zhang et al., 2001) uses a circular probe
(C-probe) in which the 3 ′ and 5 ′ ends are brought together in juxtaposition by
hybridization to a target. The two ends are then covalently linked by a T4 DNA
ligase in a target-dependent manner, producing a closed DNA circle (e.g., see
Fig. 12.2E). Upon addition of forward and reverse primers, DNA polymerase
extends the bound forward primer along the C-probe and displaces the downstream
strand generating a multimeric ssDNA. This multimeric ssDNA can then serve as a
template for reverse priming to extend and displace downstream DNA, generating a
large ramified (branching) DNA complex. This process continues until all ssDNAs
become double-stranded, resulting in an exponential amplification (Zhang et al.,
2001).
A similar procedure has also been applied to the amplification of whole genomes
for high-throughput genomic analysis (Detter et al., 2002). These procedures, however,
were inefficient in the amplification of fragmented DNA (Lage et al., 2003).
A recent adaptation termed restriction and circularization-aided RCA (RCA-RCA)
was developed for the need to amplify partially degraded DNA present in complex