In the present report, we demonstrated how complementarity between the four ribonucleotide bases in the CataCleave probe and the corresponding bases in the DNA target is required for efficient cleavage of the probe by RNase H. This specificity allows the probe to be used for the detection of SNPs, because in all our studies to date, base mismatches in this region resulted in a large reduction in the rate of probe cleavage. CataCleave probes can be designed with any sequence of four RNA bases. This lack of enzymatic sequence preference permits the versatile detection of SNPs, insertions, and deletions. The specificity of the RNase H enzyme for identifying base mismatches within its active site combined with the real-time target detection yields a novel genotyping technology.
The most important characteristics of any SNP assay are sensitivity and specificity. We rely on the active base-mismatch discriminating power of RNase H, which provides increased specificity over hybridization alone. The single-base resolving power of the CataCleave probe was demonstrated in four cases. First, probes were shown to recognize either the wild-type allele or mutant allele (C>T substitution) located in a gene coding for IGF-2. Second, in a blinded study, PCR products derived from 168 patient genomic DNA samples were genotyped for the presence of a SNP located at position 702 (C>T substitution) within the NOD2/CARD15 gene. In this experiment the CataCleave probe had a call rate of 95.2% and a genotyping accuracy of 93.7%. This call rate compares favorably with other genotyping technologies, but the accuracy rate of this study falls short of the >98% seen with other methods. There are several factors that may account for this reduced performance. One of these is the location of the R702W SNP in a region of high GC content. It was not possible to accurately genotype the 702 SNP using TaqMan SNP technology; instead, the genotyping was performed by DNA sequencing. The 702 SNP was, in fact, chosen for large scale testing with the CataCleave probe exactly because it would serve as a difficult test case. Also, we have not done any analysis to optimized the calling threshold, i.e., the real-time cleavage rate adequate for reliable discrimination. In this initial blinded study, genotypes determined for five of the 10 samples that were miscalled would have been categorized as not callable if the threshold had been increased only 30%. Removing this data would have increased the accuracy rate to 96.7%. Finally, this was the very first blinded study of CataCleave SNP genotyping. Asymmetric PCR target amplification and probe cleavage conditions may need to be adjusted to obtain more accurate results.
In the final two experiments, CataCleave probes were used to identify a 6-bp deletion in the amelogenin gene and to distinguish between a fully- and partially-methylated target DNA.
Overall, these results show that efficient probe cleavage requires correct base-pairing between the probe and the target. Base mismatches, whether caused by the presence of a SNP, frameshift, or chemical modification, result in a markedly reduced probe cleavage. The CataCleave reaction combines the stringency of hybridization with the base-resolving power of the RNase H enzyme to produce a sequence-specific target detection assay.
Target DNA detection with the CataCleave probe is a linear cyclic process. It involves multiple rounds of probe binding, RNase H mediated cleavage, and probe fragment dissociation from the same target locus. Generation of several fluorescent fragments by a single target increases detection sensitivity. This effect is shown in and under isothermal conditions. As time passes, progressively more probe is cleaved and the fluorescence signal increases because the contribution to the total intensity from each cleavage cycle is additive. Target amplification may be another way to increase the sensitivity of a genotyping assay. The CataCleave probe should be compatible with real-time genotyping methods such as PCR, strand displacement amplification (SDA), and nucleic acid sequence–based amplification (NASBA). We have previously used the CataCleave probe for the real-time detection of PCR products of the B. anthracis
Cap C gene (26
). Real-time SNP detection of PCR-amplified targets is tenable; this capability is clear in , which shows patient genomic DNA samples as amplified by PCR prior to detection.
We have evaluated additional ways to optimize the cleavage reaction. After isothermal cleavage, adherent cleaved fragments could block the target. Thus, we tested limited temperature cycling, in which brief high-temperature pulses are used to insure that all fragments dissociate. Alternatively, simply extending the detection phase of the assay creates more probe cleavage and more fluorescence. shows that a combination of these two strategies increases detection sensitivity. A two-step protocol (55°C for 50 sec and a 70°C pulse for 10 sec) was combined with increased cycle number to probe the limits of sensitivity. With sufficient time, the difference between the baseline signal and that from 1 fmol of target increases enough to suggest that even subfemtomole quantities of target can be detected. Of course, rates are also enhanced by adding more RNase H or by increasing the probe concentration.
The results of this study showed that CataCleave probes can be designed for the specific detection of SNPs (IGF-2, NOD2/CARD15), a multibase deletion (amelogenin), and partially-methylated DNA. This homogeneous genotyping method is specific, sensitive, and potentially amenable to high-throughput protocols. We are currently investigating how the probe can be used to detect SNPs associated with a variety of other disease states and we are adapting arrays of these probes for high-throughput genotyping needs.