Figure 1.
Mechanistic description of the SIBA reaction.
All single-stranded elements are coated with gp32, except for the 2′-O-methyl RNA nucleotides. Step 1: UvsX displaces gp32 on the IO and only weakly coats the primers, since they are too short for high affinity binding. Step 2: The IO invades the complementary region of the target duplex which allows partial separation of the target duplex with the downstream end still remaining double-stranded. The out-going strand of the partially separated target duplex is stabilized by gp32. Step 3: UvsX depolymerization allows the 2′-O-methyl RNA region of the IO branch migrates into the duplex. Step 4: Both the upstream and downstream region peripheral to the IO also become short enough to dissociate. Step 5: The strand displacement polymerase is able to extend the dissociated target duplex from the primers. The forward primer displaces the IO during extension of the target template. Step 8: These events lead to the production of two copies of the target duplex. The IO is released to induce further amplification.
Figure 2.
SIBA primers are unable to amplify target DNA independently of the invasion oligonucleotide (IO).
(A) Real-time monitoring of amplification using SYBR Green I, (B) melting curve analysis ((-dF (fluorescence)/dT (temperature) versus temperature), and (C) non-denaturing electrophoresis of the corresponding reaction products. Lane 1, BioRad EZ Load 20 bp Molecular Ruler (20–1000 bp); lane 2, primers + IO + template (107 copies); lane 3, primers + IO + template (105 copies); lane 4, primers + IO + water; lane 5, primers + template (107 copies); lane 6, primers + template (105 copies); lane 7, IO + template (107 copies); lane 8, primers + non-homologous IO + template (107 copies); lane 9, primers + IO + non-homologous template (107 copies); lane 10, 200 nM primers in the absence of SIBA reaction reagents; lane 11, 200 nM IO in the absence of SIBA reaction reagents; lane 12, 200 nM primers and 200 nM IO in the absence of SIBA reagents. Lanes 10–12 served as controls for monitoring the presence of oligonucleotides in the reaction products. These were diluted in TBE buffer and run alongside the SIBA reaction products. SB-F21 and SB-R21 are the forward and reverse primers, respectively. The IO used was SB-IO. The homologous target DNA used was SB-template. nhom = non-homologous to the target template (SB nhom template) or non-homologous IO (SB nhom IO).
Figure 3.
Artifactual amplification is abolished by using an invasion oligonucleotide (IO) with a 2′-O-methyl RNA modification.
(A) Configuration of the IO molecules used. (B) Real-time monitoring of SIBA reactions with SYBR Green I using different IOs: (i) IO with a 2′-O-methyl RNA modification and fully homologous to the target duplex, SB-IO; (ii) IO fully homologous to the target duplex, where the 2′-O-methyl RNA modification was replaced with natural DNA nucleotides, SB-IO DNA; (iii) IO with a 2′-O-methyl RNA modification that is not homologous to the target duplex, SB-IO DIFF-METH; and (iv) IO with the 2′-O-methyl RNA modification deleted, SB-IO NON-METH. SB-F21 and SB-R21 were the forward and reverse primers, respectively. The reactions were either performed using 106 target template molecules (SB-template) or in the absence of template.
Figure 4.
Configuration of templates used for evaluating the sensitivity of SIBA for point mutation(s).
Eight additional templates harboring different numbers of point mutation(s) 1–4 were synthesized. The point mutation(s) were either located in regions homologous to the IO DNA or the IO 2′-O-methyl RNA region (SB-IO). The black box on the templates indicates the location of the point mutation (s).
Table 1.
Sensitivity of SIBA extension to point mutations.
Figure 5.
Determination of SIBA sensitivity for Salmonella genomic DNA.
(A) Real-time monitoring of DNA amplification using SYBR Green I, (B) melting curve analysis, ((-dF (fluorescence)/dT (temperature) versus temperature) and (C) electrophoresis of the corresponding reaction products. Lane 1, BioRad EZ Load 20 bp Molecular Ruler (20–1000 bp); lanes 2–11 primers + IO + 105, 104, 103, 100, 50, 20, 10, 5, 2, or 1 copy of Salmonella genomic DNA, respectively; lane 12, primers + IO + water; lane 13, primers + IO + a mixture of non-Salmonella spp. genomic DNA (each spp. contains 1000 copies per reaction); lane 14, IO + 105 copies Salmonella genomic DNA; lane 15, primers + 105 copies Salmonella genomic DNA; 50, 20, 5 and 2 copies were omitted from Figure 5a and b for the sake of clarity. SM-F18 and SM-R16 were the forward and reverse primers, respectively. The IO used was SM-IO.