Advanced Techniques in Diagnostic Microbiology

12. Non-PCR Amplification 195
used two linear allele-specific probes each labeled with a unique fluorophore at
one end and a quencher at the other (Thomas et al., 1999; Faruqi et al., 2001;
Myakishev et al., 2001). The probe backbone contained a common binding site for
amplification-specific primer. When both primers were used in the same reaction
tube, the relative amount of each polymorphism could be determined from the
sample. This system showed a greater sensitivity than PCR SNP methods, which
was demonstrated by genotyping using 1 ng of genomic DNA (Faruqi et al., 2001).
Pickering et al. modified probe design that overcame background problems seen
by probe–probe interactions in previous studies (Faruqi et al., 2001; Pickering
et al., 2002). Thus, an end-point fluorescent assay was developed that used a highthroughput
format and did not require the use of real-time fluorescent detectors,
although the assay has been developed for real-time measurements (Christian et al.,
2001; Alsmadi et al., 2003).
Immobilized RCA has also been adapted as a signal enhancer for antibody microarrays,
or “immunoRCA” (Schweitzer et al., 2000, 2002). This assay has been
carried out in either the “sandwich” format where a matched pair of antibodies
capture are used to “sandwich” the antigen between the two antibodies (Schweitzer
et al., 2002) or in a format where the antigen itself is immobilized and a single
ligand-specific antibody is used for detection (Schweitzer et al., 2000; Zhou et al.,
2004). Secondary biotinylated antibodies, each specific for their cognate ligand,
are allowed to bind. This is followed by a third antibody specific for biotin, coupled
to an RCA primer. The RCA proceeded as in a typical amplification reaction
using fluorescent detection with Cy3 and Cy5 dyes in a microarray analysis format
(Schweitzer et al., 2002). In these initial developmental studies, up to 75 cytokines
were measured simultaneously with femtomolar sensitivity and a three log quantitative
range (Schweitzer et al., 2002).
This type of assay illustrates a number of salient points about immobilized RCA
technologies: (1) the amplicon is not diffusible, and end product inhibition typical
of PCR is not observed; therefore, sensitivity can be increased; (2) the sole reliance
on passive hybridization is eliminated; (3) universal amplification primers can be
used because the selectivity is determined by the antibody or, in the case of SNP
analysis, by the discrimination between matched and unmatched bases by DNA
ligase; (4) the technology for spotting and coupling to slides, as well as slide
reading and data analysis, has already been developed; and (5) these are end-point
assays so real-time analysis is not necessary.
RCA and In Situ Hybridization
RCA methods have been adapted to in situ hybridization, allowing an increase
in sensitivity and the ability to identify single nucleotide changes. The limits of
sensitivity of the standard fluorescence in situ hybridization (FISH) methods are in
the several kilobase range except for signal amplification using tyramide that can
lower this range to the hundreds of nucleotides (Van Tine et al., 2004). Thus, FISH
is not able to detect single nucleotide changes either in DNA within a cytological
context or in single DNA molecules (Qian and Lloyd, 2003). The application of