Probe with / restriction site
■ Figure 7-27 The first stage of SDA is the denatura-tion of the double-stranded target and annealing of primers and probes tailed with sequences including a restriction enzyme site (A; only one strand of the initial target is shown.) A second reaction (B) copies the probe, incorporating dATPaS and thereby inactivating the restriction site on the copied strand (C). This species is the target for amplification in the second stage of the reaction.
Ay Displaced , n / strand
Inactive restriction site (due to incorporation of modified nucleotides)
^ Displaced probe targets similar to LCR than PCR in that the major amplification products are the probes/primers and not the product of in vitro synthesis of target DNA. There are two stages to the SDA process. In the first stage (target generation), the target DNA is denatured by heating to 95°C. At each end of the target sequence, a primer and a probe bind close to each other (Fig. 7-27). The probes have a recognition sequence for a restriction enzyme. Exonuclease-deficient DNA polymerase derived from E. coli extends the primers, incorporating a modified nucleotide, 2'-deoxyadeno-sine 5'-O-(1-thiotriphosphate) (dATPaS). As the outer primers are extended, they displace the probes, which are also extended. A second set of complementary primers then bind to the displaced probes, and DNA polymerase extends the complementary primers, producing a double-stranded version of the probes. The probes are the target DNA for the next stage of the process.
The second stage of the reaction is the exponential probe/target amplification phase (Fig. 7-28). When the restriction enzyme is added to the double-stranded probe DNA, only one strand of the probe will be cut due to the dATPaS introduced in the extension reaction. This forms a nick in the DNA that is extended by DNA polymerase, simultaneously displacing the opposite strand. The displaced strand is also copied by primers that will restore the restriction site. As dATPaS is also used in the second-stage extension reactions, one strand of each new product will be resistant to the restriction enzyme, and the nick ing/extension reaction can repeat without denaturation. Thus, the iterative process takes place at about 52°C without temperature cycling. The product of this amplification is millions of copies of the initial probe.
This method was first widely applied to detection of M. tuberculosis.51 Methods using fluorescence polarization to detect the amplified target have been designed to test for M. tuberculosis58 and C. trachomatis.59 Addition of a fluorogenic probe to the reaction produces a fluorescent signal that corresponds to the amount of amplified target. This is the basis for the BDProbeTecET test for M. tuberculosis,60 C. trachomatis, and Neisseria gonorrhoeae.61
Qp replicase is another method for amplifying probes that have specificity for a target sequence. The method is named for the major enzyme that is used to amplify probe sequences. Qp replicase is a RNA-dependent RNA polymerase from the bacteriophage Qp.62 The target nucleic acid in this assay can be either DNA (which must first be denatured) or RNA.
The target nucleic acid is added to a well containing reporter probes. The reporter probes are RNA molecules that have specificity for the target sequence and also contain a promoter sequence (midivariant-1) that is recognized by the Qp replicase. The reporter probes are allowed to hybridize to the template. The template with
Capture probe A
■ Figure 7-28 In the second phase of SDA, the target sequence Is nicked by the restriction enzyme, generating a substrate for the polymerase, which extends the nick, displacing the opposite strand (B, D). The displaced strand is hybridized by a primer, producing another endonucleolytic target (C, E). The product of both reactions is a copy of the target with a hemisensitive restriction site (C, top). The reaction cycles of the strands are cut and extended.
bound probes is captured onto the side of the well using polyC capture probes and paramagnetic beads so that unbound reporter molecules can be washed away (Fig. 7-29). The template-probe complex is released from the polyC magnetic bead and bound to a different capture probe on a polyT paramagnetic bead. After a series of washes to remove unbound reporter probe, the templateprobe complex is again released from the magnetic bead.
For the amplification step, the probe-bound template is mixed with the Qß replicase, which replicates the probe molecules. This replication is very efficient with the gen-
Capture probe A
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