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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Clin Lab Anal. Author manuscript; available in PMC 2012 September 11.
Published in final edited form as:
PMCID: PMC3439203
NIHMSID: NIHMS396802

SNP Analysis Using CataCleave Probes

Abstract

CataCleave” probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (DNA)-ribonucleic acid (RNA)-DNA structure that can be used for real-time detection of single nucleotide polymorphisms (SNPs), insertions, and deletions. Fluorescent donor emission is normally quenched by Förster resonance energy transfer (FRET). Upon binding to the target, if the RNA/DNA hybrid is correctly base-paired, ribonuclease (RNase) H will cleave the RNA moiety and the probe fragments will dissociate. FRET is lost and the donor fluorescence signal is recovered. A single-base mismatch within the hybrid region causes probe cleavage to be significantly reduced. We designed CataCleave probes to detect SNPs located in the insulin-like growth factor 2 (IGF-2) gene and at position 702 within the NOD2/CARD15 gene. Probes were also designed to detect a six-basepair deletion in the amelogenin gene and a partially methylated target DNA. Discrimination between wild-type and SNP is demonstrated for both genes in homogeneous reactions under isothermal and temperature cycling conditions. These probes were also able to identify a multibase deletion and methylated DNA. Cleavage rates were proportional to target concentration. Probe length and position of fluorescent labels may also be modified for use in multiplexing high-throughput SNP assays. This represents a novel method for the detection of SNPs.

Keywords: fluorescence, single nucleotide polymorphism, genotype, RNase H, PCR

INTRODUCTION

Single nucleotide polymorphisms (SNPs) are single nucleotide variations in genomic deoxyribonucleic acid (DNA) that have natural occurrence. To be classified as a SNP, the least common allele must occur at a frequency of 1% or more, distinguishing SNPs from more rare point mutations. Many SNPs occur in the coding region of a gene and they can produce either neutral alterations (ones that do not alter the encoded amino acids [aa]), conservative changes (that alter aa identity but have minimal effect on protein structure or function), or nonconservative alterations that lead to dramatic effects on protein structure (usually due to frameshifts or deletions). According to the National Center for Biotechnology Institute (NCBI) SNP database, over 2.7 million unique SNPs have been identified in the human genome. Since SNPs are distributed across the entire genome, they have become important for identifying disease genes and for candidate gene association studies. In particular, several diseases have been associated with SNPs, base insertions, or deletions. These diseases include thalassemias (1), hemophilias (2), cystic fibrosis (3), Tay Sachs disease (4), Duchenne muscular dystrophy (5), and Huntington’s disease (6). The ability to detect and identify SNPs is also important in the field of pharmacogenomics, the study of how drug and dose regimens can be tailored to an individual patient. SNPs present in the MDR1 gene, for example, alter the pharmacokinetic and pharmacodynamic profiles of drugs such as digoxin, fexofenadine, and cyclosporin (7). Thus, certain treatments may benefit only a subset of patients, some may derive no benefit, and still others could experience a potentially adverse reaction. Reliable, sensitive, low-cost, and scalable technologies for SNP detection will be an important part of the medical testing arsenal for both personal and comparative genome analysis.

Many SNP genotyping technologies are available for discrimination between alternative alleles in a given DNA sample. Methods include oligonucleotide ligation (8), hybridization/annealing (molecular beacons) (9,10), primer extension (including single-base extension) (11), and enzyme cleavage (12). Some of these cleavage assays employ an enzyme with single-base specificity to precisely distinguish among products, while others rely on differences in hybridization efficiency between the probe and target. For example, the TaqMan SNP assay is performed in parallel with polymerase chain reaction (PCR) amplification (13). During the DNA synthesis phase of the PCR cycle, if there is a mismatch between the probe and the template, the number of probes that will hybridize to the template and be cleaved is lower than if there is no mismatch. A second type of enzyme cleavage assay is the Invader assay (14). This isothermal SNP assay generates a cleavable flap with the correct structure only if a signal probe matches the template and there is a simultaneous one-base invasion by the other “invader probe.”

For detection of allele-specific products several methods are currently utilized, including fluorescence detection (intensity, Förster resonance energy transfer (15), and polarization (16)), pyrosequencing (17), electrochemical detection (18), mass spectrometry (19), and atomic force microscopy (20). Nonradiative Förster resonance energy transfer (FRET) was first applied to the detection of nucleic acid sequences by Heller and Morrison (21) and their colleagues nearly 20 years ago. Since then numerous approaches for sequence-specific detection of nucleic acids have been developed and FRET has become one of the most commonly used methods, due to its low cost and ease of detection (22). The success of any SNP genotyping method relies on its specificity, sensitivity, and adaptability to high-throughput formats. Specificity can be improved by using the single-base discriminating powers of enzymes such as ribonuclease (RNase) H, DNA polymerases, or DNA ligases. For high-throughput analysis, array-based technology platforms have the advantage. These include chip-based (23), bead-based (24), and electrostringent arrays (25).

As reported recently (26), we have developed the CataCleave Probe using Cycling Probe Technology (CPT). The probe is a chimeric DNA-ribonucleic acid (RNA)-DNA sequence, in which one DNA arm is labeled with a fluorescence donor and the other is labeled with a quencher. Fluorescent donor intensity of the intact probe itself is weak due to efficient FRET. Hybridization of the probe to its complement enables RNase H to cleave the probe, disabling FRET and restoring donor fluorescence. These probes have been used for real-time detection of both PCR and rolling circle amplification (RCA) processes. Efficient cleavage of the probe requires a fully-matched target sequence. Mismatches between the RNA portion of the probe and its complementary sequence yield significant reductions in cleavage rate. This behavior allows the CataCleave probe to be used for the detection of SNPs, insertions, and deletions. In this report, CataCleave probes were designed to recognize the wild-type alleles and SNPs located within two different genes. One of the SNPs is located within an intron of the insulin-like growth factor 2 gene (IGF-2), and the other is found in the NOD2/CARD15 gene. Improper expression of IGF-2 has been associated with several human cancers, while SNPs in the NOD2/CARD15 gene are associated with an increased likelihood for the development of Crohn’s disease (CD). Probes were also designed to identify a multibase deletion in the amelogenin gene, useful in primate sex determination, and to see if the probe could differentiate between fully- and partially-methylated target DNA after treatment with sodium bisulfite. The results below demonstrate that CataCleave probes are useful for SNP genotyping and the identification of base insertions, deletions, and partially methylated DNA.

MATERIALS AND METHODS

Thermostable RNase H was purchased from Epicentre Biotechnologies (Madison, WI). All DNA synthesis reagents were purchased from Glen Research. Taq DNA polymerase was purchased from New England Biolabs (Ipswich, MA).

Preparation of Oligonucleotides and CataCleave Probes

Oligonucleotides and CataCleave probes were synthesized using a PerSeptive Biosystems Expedite nucleic acid synthesis system (Applied Biosystems, Foster, CA) and purified as previously described (26). Nucleic acid sequences are listed in Table 1.

TABLE 1
Sequences for IGF-2, Crohńs 702, amelogenin- and methylation-specific CataCleave probes, target DNAs, and PCR primers*

RNase H Cleavage Reactions

Reactions were performed in RNase H cleavage buffer containing 10 mM MgCl2, 10 mM KCl, 0.1% Triton X-100, and 20 mM Tris-HCl, pH 8.0, in a Bio-Rad iCycler (Bio-Rad, Hercules, CA).

PCR Reactions

Reactions were carried out in Taq polymerase buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100) containing 3 mM MgCl2, 400 μM deoxynucleoside triphosphates, 50 pmol of R702W-F primer, 5 pmol of R702W-R primer, 1 μL diluted NOD2/CARD15 PCR product, and 2.5U of Taq DNA polymerase in a total volume of 50 μL. The protocol consisted of 1 cycle at 95°C for 3 min, followed by a 50-cycle amplification (95°C for 30 sec, 50°C for 45 sec, and 72°C for 45 sec), and a final extension step at 72°C for 7 min.

Bisulfite Treatment of DNA

A solution containing denatured target DNA was made by dissolving 0.1 nmol of DNA in 50 μL of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM ethylene diamine tetraacetic acid [EDTA]) and adding 5 μL of 3 M NaOH (final concentration 0.3 M). This mixture was incubated at 37°C for 10 min. To this solution, 510 μL of 40.5% sodium bisulfite (final concentration 3.3 M), 30 μL of 10 mM hydroquinone, and 15 μL of H2O were added. This mixture was incubated in the dark for 16 hr at 55°C. The DNA was then purified by ethanol precipitation and resuspended in 50 μL of TE. Following purification, 5 μL of 3 M NaOH was added to the sample and the solution was incubated at 37°C for 15 min. The solution was neutralized by adding ammonium acetate (pH 7.0) to 3 M and the DNA was precipitated with ethanol, washed twice with 70% ethanol, and dried.

RESULTS

SNP Analysis of IGF-2 Using CataCleave Probes

RNase H recognizes and cleaves the RNA sequences within a double-stranded probe/target-DNA hybrid, enabling CPT (27). In general, a CPT probe contains four contiguous RNA bases flanked on each side by DNA arms. It has been reported that RNase H cleavage requires at least four ribonucleotides and that the preferred cleavage position is located 5′ to the penultimate ribonucleotide (28).

We developed fluorescent CataCleave probes to directly monitor RNase H reactions, measuring fluorescence intensity without physically separating the cleaved probes (29). We previously observed a significant reduction in cleavage rates when target DNA contained single base mismatches within the RNA/DNA hybridizing region (26). Hence, we prepared a CataCleave probe for a SNP located within intron 2 of IGF-2 (Table 1). The gene for IGF-2 is located on chromosome 15 and encodes a regulatory peptide critical for normal fetal growth. Numerous SNPs have been identified throughout the gene, with several located within introns. One of these, rs734351, has been identified in the National Cancer Institute “SNP500” cancer database as being important in the molecular epidemiology of cancer. Improper expression of IGF-2 has been implicated in tumor progression in a variety of human neoplasms, including neuroblastomas (30) and breast cancer (31).

CataCleave probes for rs734351 wild-type and mutant alleles were designed so that both fluorescein (FRET donor) and tetramethylrhodamine (FRET acceptor) were attached internally to the probe (26). Cleavage of the wild-type probe IGF-2PW was initially tested with its target, IGF-2TW (Table 1). A total of five different concentrations of IGF-2TW were initially incubated at 55°C in cleavage buffer with 20 pmol of probe IGF-2PW. Following preincubation, RNase H was added and donor fluorescence was monitored for enhancement (Fig. 1A). As the concentration of target decreased, the cleavage rate decreased in a dose-dependent fashion. A mutant target (IGF-2TM, Table 1), with a SNP in the RNA region (5′-TTCT-3′ changes to 5′-TTTT-3′, which is specific for the mutant allele (C>T)) was also tested. No significant cleavage was detected with varying concentrations of mutant target DNA (Fig. 1B).

Fig. 1
Specificity and concentration dependent cleavage of IGF-2PW CataCleave probe in the presence of IGF-2TW. A: Cleavage reactions were performed at 55°C with either 5 pmol ([diamond]), 2 pmol (■), 1 pmol (▲), 0.5 pmol (●), ...

For completeness, we prepared a “mutant sensing” probe (IGF-2PM, Table 1). When IGF-2PM was incubated with different concentrations of mutant target, dose-dependent cleavage was observed (Fig. 2A). As expected, when mutant probe IGF-2PM was incubated with wild-type target IGF-2TW, no significant cleavage was observed (Fig. 2B). These combined results demonstrate the single-base specificity of these CataCleave Probes.

Fig. 2
Specificity and concentration-dependent cleavage of IGF-2PM CataCleave probe in the presence of IGF-2TM. A: Cleavage reactions were performed at 55°C with either 5 pmol ([diamond]), 2 pmol (■), 1 pmol (▲), 0.5 pmol (●), ...

Flanking Hybridized Regions and Their Impact on Cleavage Efficiency

Previous studies on the kinetics of RNase H (28) showed that the cleavage rate with three, rather than four, ribonucleotides was greatly diminished, placing stringent limits on the size of the RNA portion of the probe. Prior studies did not vary the length of the flanking DNA “arms.” The IGF-2PM probe is a 24-mer with two 10-base DNA arms, and we have prepared two additional target sequences with shorter arms. The target IGF-2T18M is a 18-mer with two 7-nucleotide arms, and IGF-2T16M is a 16-mer with two 6-nucleotide arms (Table 1). One consequence of shortening the target is a reduction in the melting temperature of the probe/target complex. Cleavage efficiency experiments were performed at three temperatures, 55°C, 50°C, and 45°C. A total of 20 pmol of IGF-2PM was incubated with 5 pmol each of four target DNAs in RNase H buffer. In addition to the three different lengths of target DNAs, the 24-mer IGF-2TW was used as a negative control.

Figure 3A shows that the mutant probe was efficiently cleaved in complexes with all three mutant target DNAs. Furthermore, the probe incubated with the longer IGF-2TM template showed a higher cleavage efficiency compared to shorter targets at 55°C. These differences are likely due to the melting temperature of each probe/target matching the reaction temperature. Efficient cyclic cleavage requires repeated annealing, cleavage of the RNA sequence, and dissociation of the cleaved probes. The shorter target sequences (with lower melting temperatures) will not anneal efficiently at elevated temperatures. Lower temperatures improve the cleavage rate with these short targets. Figure 3C shows that the shortest target displayed a higher cleavage rate at 45°C. Conversely, longer targets may not efficiently dissociate at reduced temperatures. At 50°C, the cleavage rates with all three targets were similar (Fig. 3B). Overall, changing arm length plays a role in promoting efficient cyclic cleavage by modifying the complex melting temperature.

Fig. 3
Effects of DNA arm length on the efficiency of IGF-2PM CataCleave probe cleavage. A: Cleavage reactions were performed at 55°C with either 5 pmol of IGF-2T16M ([diamond]), 5 pmol of IGF-2T18M (□), or 5 pmol of IGF-2TM (▲) in 20 ...

Effect of Temperature Cycling on Cleavage Efficiency

If multiple probes unique for different targets are to be used under the same reaction conditions, as in a microarray application, adjusting the length of each probe for efficient cleavage at the same temperature may be inefficient; It is more convenient to adjust the reaction temperature. As an alternative, temperature cycling can be incorporated to “sweep” a range of melting temperatures across different complexes. The thermostable RNase H used in the reactions is active over a temperature range of at least 45°C to 70°C, permitting such cycling. Another advantage is that a high-temperature pulse can be incorporated into each cycle to ensure complete dissociation of probe halves. Otherwise, residual bound probe fragments could block some target sites. In Fig. 3C, cleavage was performed at 45°C. The same reaction mixtures were also processed using a two-step temperature cycling protocol (45°C for 55 sec followed by 70°C for 5 sec) and the results are presented in Fig. 4. The initial velocities for all three reactions in Fig. 4 are not only faster, but also more closely matched. The reactions in Fig. 3C never reach a steady-state level, signifying that not all of the available CataCleave probe was consumed. In Fig. 4, the relative fluorescence intensities for all three datasets plateau after approximately 40 cycles, indicating that most of the probe has been cleaved. These results demonstrate that temperature cycling can be used to increase the rate and efficiency of CataCleave reactions involving different length probes by: 1) providing each probe/target complex exposure to an optimal reaction temperature at some time; and 2) promoting full product dissociation with a high temperature pulse.

Fig. 4
Effects of temperature cycling on the efficiency of IGF-2PM CataCleave probe cleavage. Each temperature cycle consisted of two steps; 55 sec at 45°C and 5 at 70°C. Real-time data were collected during the 45°C step. Reactions contained ...

SNP Analysis of NOD2/CARD15 Gene Using CataCleave Probes

SNPs within the NOD2/CARD 15 gene have been found to correlate with an increased likelihood for the development of CD, but not for ulcerative colitis (32). CD is a chronic, heterogeneous, transmural inflammatory bowel disease (IBD) that can involve any portion of the gastrointestinal tract, most often the distal ileum. There is evidence of phenotypic heterogeneity within CD as demonstrated by variations in age at onset, site of disease involvement, and disease behavior. A family history of IBD has been associated with early onset disease and ileal disease location (33). The associations of three coding SNPs, R702W, G908R, and L1007fsinsC in the NOD2/CARD15 gene located on chromosome 16 have been clearly established to increase susceptibility to CD (34).

The 702 SNP is located near a domain coding for a leucine-rich repeat region that binds to muramyl dipeptide, a component of bacterial cell wall peptidoglycan. The R702W change results in markedly decreased ability of NOD2/CARD15 to activate nuclear factor kappa B in response to muramyl dipeptide (35). Like the IGF-2 mutation, this SNP also involves a C>T base change. In comparison to the sequence of the IGF-2 target, that for the R702 sequence contains a higher GC content. Because of this, 24-mer probes designed to recognize the R702W alleles would have very high melting temperatures and may not promote efficient cleavage reactions. Based on previous results, we designed two R702W CataCleave probes as 16-mers having two 6-base DNA arms along with the required four ribonucleotide identification regions. Another difference in the design of the probes was that the fluorescein and carboxytetramethylrhodamine (TAM-RA) labels were located at the 5′ and 3′ ends of each probe instead of internally. This simplified probe synthesis, while maintaining efficient FRET because the donor-quencher distance remained less than R0 for the pair.

Cleavage of the wild-type sensing probe (R702PW) and mutant sensing probe (R702WPM), was initially examined with their corresponding target DNAs (the wild-type target R702TW and the mutant target R702WTM) (Table 1). Incubation of either the wild-type probe with wild-type target or mutant probe with mutant target resulted in efficient probe cleavage. As expected, incubation of the wild-type probe with mutant target, or mutant probe with wild-type target resulted in significantly reduced probe cleavage (data not shown). We also examined the detection sensitivity of the wild-type R702PW probe in a temperature cycling environment using six different concentrations of wild-type R702TW target. A two-step temperature cycling protocol (50 sec at 55°C and 10 sec at 70°C) was chosen over isothermal incubation for this and subsequent experiments. As shown in Fig. 5, enhancement of donor fluorescence over the negative control was observed in all cases. With 2 pmol of target, donor fluorescence rapidly increased. As the concentration of R702TW decreased, the cleavage rate decreased in a dose-dependent matter. Using this protocol, 1 fmol of target could be easily differentiated from the background after 50 cycles.

Fig. 5
Concentration dependent cleavage using R702PW. Reactions contained either 2 pmol ([big up triangle, open]), 1 pmol (□), 0.5 pmol (○), 0.1 pmol (▲), 0.05 pmol (■), or 1 fmol (●) of R702TW in 20 μL of cleavage buffer containing ...

Genotyping R702W NOD2/CARD15 Gene PCR Products Generated From Genomic DNA

In the experiments above, the target DNA was a synthetic single-stranded DNA. Clinical SNP genotyping studies are normally performed with either double-stranded genomic DNA, or duplex DNA generated by a nucleic acid amplification process like PCR. With a denatured double-stranded substrate, there is a competition between CataCleave probe binding to the target strand and reannealing to reform the original duplex. Higher quality target for the CataCleave probe reaction can be obtained with asymmetric PCR, in which one of the primers is used in large excess over the other. During the early cycles of PCR the concentration of both strands increases exponentially, while in later cycles only the strand complimentary to the primer in excess is amplified. The primer chosen to be in excess is the one that will generate target complimentary to the probe. This procedure produces mostly single-stranded DNA that is an ideal target for analysis. We sought to genotype DNA from three different individuals homozygous for the wild-type allele, heterozygous or homozygous for the R702W SNP. Previously genotyped genomic DNA samples (36) were amplified by PCR using 50 pmol of primer R702W-F and 5 pmol of primer R702W-R (Table 1) in PCR buffer to generate a 511-bp PCR product. Separate cleavage reactions were performed using 10 μL of each PCR product, along with 5 pmol of either R702PW or R702WPM in cleavage buffer containing RNase H. Prior to addition of RNase H the samples were denatured at 95°C for 90 sec. A two-step thermal cycling protocol was used (50 sec at 55°C and 10 sec at 70°C), with real-time data collected during the 55°C step. Individual positive control reactions were also run using 5 pmol of each probe and its fully complementary target. In Fig. 6A, reactions were performed with a PCR sample from an individual homozygous for the wild-type allele. An enhancement of donor fluorescence can be seen for the wild-type sensing R702PW probe, while emission from the SNP sensing R702WPM probe remained unchanged. In Fig. 6B, reactions were performed with a PCR sample from a heterozygous individual and an enhancement of donor fluorescence is observed for both the wild-type and SNP sensing probes. In Fig. 6C, reactions were performed with a PCR sample from an individual homozygous for the mutant allele. In this example, fluorescence from the R702WPM probe is enhanced, while that from the R702PW probe remained virtually unchanged. This experiment is an example of successful SNP genotyping. Naturally, this technology needs to be validated in a large-scale, blinded study.

Fig. 6
SNP genotyping of 702 NOD2/CARD15 gene PCR products. Cleavage reactions contained 10 μL of PCR product and either 5 pmol (▲) of R702WPM or 5 pmol (●) of R702PW, and 2.5U of thermostable RNase H in 20 μL of cleavage buffer. ...

Such a study was performed using genomic DNA samples from 168 patients participating in a IBD genotyping survey at Johns Hopkins University (JHU) Medical Center in Baltimore, MD. The 702 genotype for all patient samples was initially determined by DNA sequencing and the results were not released by JHU prior to CataCleave genotyping. To maintain patient confidentiality, the samples were coded by number and we were not allowed to remove genomic DNA samples from the laboratory at JHU. For this reason, genomic DNA samples were first amplified by conventional PCR using equimolar concentrations of primers R702W-F and R702W-R and the PCR product was removed for genotyping. Target was generated for genotyping by asymmetric PCR and cleavage reactions were performed using the same conditions as in the above examples (Fig. 6). Out of the 168 patient samples, 160 generated signals with one or both probes greater than the negative control signal, and we judged these to have sufficient target for genotyping (see Discussion). The call rate was therefore 95.2% (160/168). Of the remaining 160 samples, the genotype determined by CataCleave probe matched the sequencing data for 150 of the samples, for an accuracy rate of 93.7% (150/160).

Sex Determination Based on a Deletion in the Amelogenin Gene

Beyond SNPs, nucleic acid polymorphisms may also involve insertions or deletions in DNA. One example of this is in primate sex determination. The human amelogenin gene is located on both the X and Y chromosome in single copies (37) and has been used for sex determination (38). PCR primers are designed to flank a 6-bp deletion within intron 1 of the X chromosome gene. The resulting PCR product is 106 bp, while the product from the Y chromosome is 112 bp. A comparison of the proportion of PCR products from the X and Y chromosomes equals their initial ratio in the DNA sample. CataCleave probes were designed to recognize a portion either the X (AMELX-P) or Y (AMELY-P) chromosomal PCR product. In addition, targets complimentary to the probes (AMELX-T and AMELY-T) were also synthesized (Table 1). Cleavage specificity of the X and Y chromosomal PCR product sensing probes was tested using targets in all possible combinations. In each case 5 pmol of probe was incubated at 55°C in cleavage buffer with 2 pmol of target. The results in Fig. 7 show that only fully complimentary target catalyzes the efficient cleavage of the corresponding probe (AMELX-P + AMELX-T and AMELY-P + AMELY-T), while a mismatched set results in greatly reduced cleavage (AMELX-P + AMELY-T and AMELY-P + AMELX-T). These results show that the CataCleave probe can also be used to detect genomic DNA deletions.

Fig. 7
Sequence-specific cleavage of CataCleave probes for X and Y amelogenin targets. Cleavage reactions were performed at 55°C and contained either 2 pmol of AMELY-T and 5 pmol of AMELY-P ([big up triangle, open]), 2 pmol of AMELX-T and 5 pmol of AMELX-P (▲), ...

Detection of a Partially-Methylated Target DNA Using CataCleave Probe

CataCleave probe was also used in an experiment to identify a partially-methylated target DNA. DNA methyltransferases transfer a methyl group to the 5-carbon position of specific cytosines after replication. This modification occurs predominately on cytosines located 5′ to guanosine, the so-called CpG dinucleotides. These dinucleotides are overrepresented in regions of DNA called “islands,” which are associated with promoters of about half of all genes (39). Aberrant DNA methylation is commonly observed in cancer cells and several methods to detect changes in methylation state have been developed (40). These methods exploit the ability of sodium bisulfite to convert unmethylated cytosine to uracil. This conversion is operationally similar to the introduction of a C>T SNP in the target. An experiment was performed to determine if the CataCleave probe can be used to identify the methylation state of cytosine in a CpG dinucleotide. The target chosen for this experiment also includes a SNP associated with hereditary hearing loss that had been studied previously (data not shown). The 167MT target (Table 1) was synthesized with 5-methylcytosine replacing cytosine in the sequence, while the 167MT-ME target was synthesized with 5-methylcytosine in all locations except for the third cytosine in the sequence “5′-CCCG.” These four bases lie opposite to their ribonucleotide counterparts when hybridized to the 167PM probe. Both targets were treated with sodium bisulfite and tested for their ability to catalyze cleavage of the probe. Specificity was tested by incubating 5 pmol of 167PM probe with either 0.1 pmol of fully-methylated (167MT) or partially-methylated (167MT-ME) target at 55°C in cleavage buffer. Figure 8 shows that the fully-methylated target continues to catalyze cleavage of the probe, while the cleavage rate with the partially-methylated target is greatly reduced. These results are to be expected, since bisulfite treatment does not change the identity of any of the bases in the 167MT sequence, but the third cytosine within the “5′-CCCG” sequence of the 167MT-ME target is converted to a “U.” The resulting base mismatch with the probe leads to a reduced cleavage rate, just as is observed with a SNP. This experiment demonstrates that the CataCleave probe may also be useful in detecting methylated DNA.

Fig. 8
Identification of partially methylated DNA target. 167MT was synthesized with 5-methylcytosine in all positions and 167MT-ME was synthesized with 5-methylcytosine in all positions except number eight in the sequence. Both targets were treated with sodium ...

DISCUSSION

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 Figs. 1A and and2A2A 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 Fig. 6, 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. Figure 5 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.

Acknowledgments

Research supported in part by the Department of Intramural Research, NHLBI. No endorsement of products by the U.S. Government is expressed or impaired.

Abbreviations

aa
amino acid
FAM
carboxyfluorescein
HEX
hexachlorofluorescein
TAMRA
carboxytetramethylrhodamine
Tris-HCl
Tris (hydroxymethyl) aminomethane hydrochloride
Taq
Thermus aquaticus
RCA
rolling circle amplification
SDA
strand displacement amplification
NASBA
nucleic acid sequence–based amplification

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