DNA mutation detection with chip-based temperature gradient capillary electrophoresis using a slantwise radiative heating system

A simple and robust chip-based temperature gradient capillary electrophoresis (TGCE) system was developed for DNA mutation/single-nucleotide polymorphism (SNP) analysis using a radiative heating system. Reproducible, stable and uniform temperature gradients were established along a 3 cm length of the electrophoretic separation channel using a single thermostated aluminium heater plate. The heater was slightly slanted relative to the plane of the glass chip at 0.2-1.3 degrees by inserting thin spacers between the plate and chip at one end to produce differences in radiative heating that created the temperature gradient. On-chip TGCE analyses of 4 mutant DNA model samples amplified from plasmid templates, each containing a single base substitution, with a wide range of melting temperatures, showed that mutations were successfully detected under a wide temperature gradient of 10 degrees C and within a short gradient region of about 3 cm (3.3 degrees C cm(-1) gradient). The radiative heating system was able to establish stable spatial temperature gradients along short microfluidic separation channels using simple peripheral equipment and manipulation while ensuring good resolution for detecting a wide range of mutations. Effectiveness of the system was demonstrated by the successful detection of K-ras gene mutations in 6 colon cancer cell lines.     

Detection of single nucleotide polymorphisms by the specific interaction between transition metal ions and mismatched base pairs in duplex DNA

Single nucleotide polymorphisms (SNPs) are base differences in the human genome. These differences are favorable markers for genetic factors including those associated with risks of complex diseases and individual responses to drugs. When two duplex DNAs with different types of SNPs are mixed and reannealed, the two novel heteroduplexes containing mismatched base pairs are formed in addition to the two initial perfectly matched homoduplexes. Heteroduplex analysis recognizing the newly formed mismatched base pairs is useful for SNP detection. Various strategies to detect the mismatched base pairs were devised due to the potential applications of SNPs. However, they were not always convenient and accurate. Here, we propose a novel strategy to detect the mismatched base pairs by the specific interaction between the Hg²⁺ ion and a T:T mismatched base pair and that between the Ag⁺ ion and a C:C mismatched base pair. UV melting indicated that the melting temperature of only the heteroduplexes with the T:T and C:C mismatched base pair specifically increased on adding the Hg²⁺ and Ag⁺ ion, respectively. Fluorescence resonance energy transfer analyses indicated that the intensity of fluorophore emission of only the fluorophore and quencher-labeled heteroduplexes with the T:T and C:C mismatched base pair specifically decreased on adding the Hg²⁺ and Ag⁺ ion, respectively. We propose that the addition of the metal ion could be a convenient and accurate strategy to detect the mismatched base pair in the heteroduplex. This novel strategy might make the heteroduplex analysis easy and eventually lead to better SNP detection.     

变性高效液相色谱法在α-血红蛋白稳定蛋白基因检测中的应用

建立了采用变性高效液相色谱(DHPLC)对α-血红蛋白稳定蛋白(AHSP)基因进行基因分型和突变筛查的新方法。将AHSP基因序列分成6个片段,因第一、二、四、六个片段均含有1～2个常见单核苷酸多态性(single nucleotide polymorphism, SNP)位点,需单个标本分别进行检测;第三、五个片段不含有常见SNP位点,采用DHPLC结合DNA(脱氧核糖核酸)池的方法进行检测。以基因测序为金标准对所建立的AHSP基因检测方法进行方法学评价,结果显示: 40个样品的DHPLC检测结果与测序结果之间完全吻合,说明所建立的检测方法能对AHSP基因6种常见SNP进行准确基因分型。应用DHPLC对365个样品的AHSP基因进行检测,发现2个罕见SNP(11810 G＞A和12802 C＞T);同时还发现2个错义突变(AHSP D29V和AHSP V56G), AHSP D29V突变为新突变,AHSP V56G为罕见突变。结果表明采用DHPLC法可有效地对AHSP基因进行基因分型和突变筛查。     

Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.     

Sensing mispaired thymines in DNA heteroduplexes using an electroactive osmium marker: towards electrochemical SNP probing

A complex OsO₄, 2,2′-bipyridine (Os,bipy), has been used for electroactive labeling of biopolymers as well as for probing of nucleic acids and protein structure and interactions. In DNA, Os,bipy forms electrochemically active adducts with pyrimidine nucleobases, exhibiting highly selective modification of thymine residues in single-stranded DNA. Here, we show that modification of rare thymine residues (one thymine among several tens of unreactive purine bases) can easily be detected by means of a simple ex situ voltammetric analysis using carbon electrodes. Based on this remarkable sensitivity of detection, Os,bipy has been used as an electroactive probe for unpaired and/or mismatched thymine residues within DNA heteroduplexes. Site-specific chemical modification of the DNA with the Os,bipy has allowed a clear distinction between perfectly base-paired DNA homoduplexes and mismatched heteroduplexes, as well as discrimination among heteroduplexes containing one or two mispaired thymines, a single thymine insertion, or combination of a mispair and an insertion.     

Parallel optimization and genotyping of multiple single-nucleotide polymorphism markers by sample pooling approach using cycling-gradient CE with multiple injections

Increasing importance of single-nucleotide polymorphisms (SNPs) in determination of disease susceptibility or in prediction of therapy response brings attention of many molecular diagnostic laboratories to simple and low-cost SNP genotyping methodologies. We have recently introduced a mutation detection technique based on analysis of homo- and heteroduplex PCR fragments resolved in cycling temperature gradient conditions on a conventional multicapillary-array DNA sequencer. The main advantage of this technique is in its simplicity with no requirement for sample cleanup prior to the analysis. In this report we present a practical application of the technology for genotyping of SNP markers in two separate clinical projects resulting in a combined set of 44 markers screened in over 500 patients. Initially, a design of PCR primers and conditions was performed for each SNP marker. Then, optimization of CE running conditions (limited just to the proper selection of temperature cycling) was performed on pools of 20 DNA samples to increase the probability of having each of the two allele types represented in the sample. After selecting the optimum conditions, screening of markers in patients was performed using a multiple-injection approach for further acceleration of the sample throughput. The rate of successful optimization of experimental conditions without any pre-selection based on the SNP sequence or melting characteristics was 80% from the initial SNP marker candidates. By studying the failed markers, we attempt to identify critical factors enabling successful typing. The presented technique is very useful for low to medium sized SNP genotyping projects mostly applied in pharmacogenomic research as well as in clinical diagnostics. The main advantages include low cost, simple setup and validation of SNP markers.     

Review of denaturant capillary electrophoresis in DNA variation analysis

Analyses of germline and somatic single-nucleotide DNA variations are important in both population genetics research and clinical practice. Reliable and inexpensive methods that are flexible and designed for automation are required for these analyses. Present day DNA sequencing technology is too expensive for testing all 22-25 000 human genes in populations genetics studies or in scanning large numbers of tumors for novel mutations. Denaturant capillary electrophoresis (DCE) has the potential to meet the need for large-scale analysis of DNA variants. Several different analyses can be performed by DCE, including mutation analysis, single-nucleotide polymorphism (SNP) discovery in individual and pooled samples, detection of allelic imbalance, and determination of microhaplotypes. Here we review the theoretical background of the method, its sensitivity, specificity, detection limit, throughput, and repeatability in the light of current literature in the field.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

A SNaPshot assay for genotyping 44 individual identification single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs), which have relatively low mutation rates and can be genotyped after PCR with shorter amplicons compared with short tandem repeats (STRs), are being considered as potentially useful markers in forensic DNA analysis. Those SNPs with high heterozygosity and low Fst (F-statistics) in human populations are described as individual identification SNPs, which perform the same function as STRs used in forensic routine work. In the present study, we developed a multiplex typing method for analyzing 44 selected individual identification SNPs simultaneously by using multiplex PCR reaction in association with fluorescent labeled single base extension (SBE) technique. PCR primers were designed and the lengths of the amplicons ranged from 69 to 125?bp. The population genetics data of 79 unrelated Chinese individuals for the 44 SNP loci were investigated and a series of experiments were performed to validate the characteristic of the SNP multiplex typing assay, such as sensitivity, species specificity and the performance in paternity testing and analysis of highly degraded samples. The results showed that the 44-SNPs multiplex typing assay could be applied in forensic routine work and provide supplementary data when STRs analysis was partial or failed.     

Intramolecular controls for the generation of clinical-quality SNP genotypes and the assessment of normal heterozygote imbalance

We present an inventive method that generates intramolecular controls for SNP analysis, termed Mirror SNPs. Using the ovine diseases of callipyge and scrapie as examples, we describe the PCR-driven production of balanced heterozygote copies of the various SNPs implicated in these diseases. In the absence of a callipyge-positive control DNA, we generated a balanced heterozygote Mirror SNP that represents both the wild-type and mutant forms of the causal polymorphism. Simultaneous analysis of this artificial Mirror SNP and the Real (target) SNP was used to prove the absence of the mutant form of the nucleotide at the Real SNP position in tested samples. Scrapie susceptibility was assessed using a PCR-driven system which generated four separate Mirror SNPs, and these enabled the confirmation of an apparent departure from 'balanced' heterozygote appearance at Real SNPs tested. Mirror SNP technology is generic and will enable the accurate assessment of rare and medically important SNP variants, more accurate frequency determinations, and the potential assessment of SNPs in 'mixed template' samples common in forensic analyses.     

An allelic ladder based upon reference alleles for mtDNA SNP analysis using the SNaPshot technique

The analysis of mitochondrial DNA (mtDNA) single-nucleotide polymorphisms (SNPs) using the SNaPshot technique (Applied Biosystems) is a fast and sensitive method for the reliable identification of disease-associated mtDNA SNPs, genetic ancestry mtDNA SNPs and forensically important mtDNA SNPs. The detection of many SNPs in one multiplex PCR and one subsequent multiplex minisequencing reaction is challenging for laboratories who want to establish this technique, due to the problem that there is no allelic ladder available for mtDNA SNP analysis via SNaPshot technique. Normally, the laboratory has to invent long-term testing and studies. The interpretation of false and correct alleles is up to some specialists knowing the expected and the estimated size of each allele SNP. We here present a protocol to assemble up to 84 alleles of 42 different mtDNA SNPs in an allelic ladder that is based upon reference alleles. We recommend using allelic ladders/reference alleles for SNP analysis to maintain high-quality analysis standards.     

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pyrosequencing‐based strategy for a successful SNP detection in two hypervariable regions: HV‐I/HV‐II of the human mitochondrial displacement loop

Forensic analysis of mitochondrial displacement loop (D‐loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high‐throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D‐loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D‐loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.     

Application of SNPs with low minor allele frequencies in missing person identification (MPI) through kinship analysis of DNA mixtures

The need to identify a missing person (MP) through kinship analysis of DNA samples found at a crime scene has become increasingly prevalent. DNA samples from MPs can be severely degraded, contain little DNA and mixed with other contributors, which often makes it difficult to apply conventional methods in practice. This study developed a massively parallel sequencing–based panel that contains 1661 single-nucleotide polymorphisms (SNPs) with low minor allele frequencies (MAFs) (averaged at 0.0613) in the Chinese Han population, and the strategy for relationship inference from DNA mixtures comprising different numbers of contributors (NOCs) and of varying allele dropout probabilities. Based on the simulated dataset and genotyping results of 42 artificial DNA mixtures (NOC = 2–4), it was observed that the present SNP panel was sufficient for balanced mixtures when referenced to the closest relatives (parents/offspring and full siblings). When the mixture profiles suffered from dropout, incorrect assignments were markedly associated with relatedness, NOC and the dropout level. We, therefore, indicate that SNPs with low MAFs could be reliably interpreted for MP identification through the kinship analysis of complex DNA mixtures. Further studies should be extended to more possible scenarios to test the feasibility of this present approach.     

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.     

A new set of DIP‐SNP markers for detection of unbalanced and degraded DNA mixtures

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP‐SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP‐SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP‐SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP‐SNPs in a Chinese Han population. The DIP‐SNPs were capable of detecting the minor contributor's allele in home‐made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 –10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP‐SNPs may serve as a valuable tool in detection of UDM in forensic medicine.     

The potential of electrophoretic mobility shift assays for clinical mutation detection

As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.     

A fluorescence light-up Ag nanocluster probe that discriminates single-nucleotide variants by emission color

Rapid and precise screening of small genetic variations, such as single-nucleotide polymorphisms (SNPs), among an individual's genome is still an unmet challenge at point-of-care settings. One crucial step toward this goal is the development of discrimination probes that require no enzymatic reaction and are easy to use. Here we report a new type of fluorescent molecular probe, termed a chameleon NanoCluster Beacon (cNCB), that lights up into different colors upon binding SNP targets. NanoCluster Beacons (NCBs) are collections of a small number of Ag atoms templated on single-stranded DNA that fluoresce strongly when placed in proximity to particular DNA sequences, termed enhancers. Here we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. Chameleon NCBs exploit this color shift to directly detect SNPs, based on the fact that different SNPs produce a different alignment between the Ag nanocluster and the enhancer. This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Samples with single-nucleotide variations can be easily identified by the naked eye under UV excitation, making this method a reliable and low-cost assay with a simple readout format.     

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.     

Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.