Single Base Pair Mismatch Research Articles

Constitutional mismatch repair-deficiency syndrome

The mismatch repair (MMR) machinery contributes to genome integrity and the MLH1 , MSH2 , MSH6 and PMS2 genes play a crucial role in this process. MMR corrects single base-pair mismatches and small insertion-deletion loops that arise during replication. Moreover, the MMR system is involved in the

An essential DNA strand-exchange activity is conserved in the divergent N-termini of BLM orthologs

The gene mutated in Bloom's syndrome, BLM, encodes a member of the RecQ family of DNA helicases that is needed to suppress genome instability and cancer predisposition. BLM is highly conserved and all BLM orthologs, including budding yeast Sgs1, have a large N-terminus that binds Top3-Rmi1 but has no known catalytic activity. In this study, we describe a sub-domain of the Sgs1 N-terminus that shows in vitro single-strand DNA (ssDNA) binding, ssDNA annealing and strand-exchange (SE) activities. These activities are conserved in the human and Drosophila orthologs. SE between duplex DNA and homologous ssDNA requires no cofactors and is inhibited by a single mismatched base pair. The SE domain of Sgs1 is required in vivo for the suppression of hyper-recombination, suppression of synthetic lethality and heteroduplex rejection. The top3Delta slow-growth phenotype is also SE dependent. Surprisingly, the highly divergent human SE domain functions in yeast. This work identifies SE as a new molecular function of BLM/Sgs1, and we propose that at least one role of SE is to mediate the strand-passage events catalysed by Top3-Rmi1.

The Rule of Seven Revealed by Observing DNA Annealing in a Nanocontainer

Although the melting temperature (Tm) of DNA can be predicted with great accuracy, little is understood about the basic rates governing the helix-coil transitions between two strands of DNA. Here we adapt a porous vesicle encapsulation method with single-molecule fluorescence to measure these rates directly for a 9 bp DNA duplex (Tm=23°C) and characterize their variation with mismatched basepairs. A single basepair mismatch can cause up to three orders of magnitude variation in duplex stability. Surprisingly, we found that the rate of DNA annealing shows an abrupt 100 fold change depending on whether there are 7 or more contiguous bp or not (∼106 M−1 sec−1 vs. ∼104 M−1 sec−1). Similar results were obtained for a microRNA seed with 7 bp match to p53 and 6 bp match to LIN28 gene sequences. Our results suggest a phenomenological cooperativity of 7 basepairs during Watson-Crick sequence recognition, with fundamental implications in nucleic acid pairing processes such as microRNA targeting and silencing in posttranscriptional regulation, and have practical implications for DNA microarray applications.

Detection of single nucleotide polymorphisms using a DNA Holliday junction nanoswitch—a high-throughput fluorescence lifetime assay

We report a simple DNA sensor device, using a combination of binding and conformational switching, capable of rapid detection of specific single nucleotide polymorphisms in an unlabelled nucleic acid target sequence. The detection is demonstrated using fluorescence lifetime measurements in a high-throughput micro plate reader instrument based on the time-correlated single-photon counting technique. The sensor design and instrumental architecture are capable of detecting perturbations in the molecular structure of the probe-target complex (which is similar to that of a Holliday junction), due to a single base pair mismatch in a synthetic target. Structural information, including fluorophore separations, is obtained using time-resolved Förster resonance energy transfer between two fluorophores covalently bound to the probe molecule. The two probes required are designed to detect a single nucleotide polymorphism from a sequence present on each of the two copies of human chromosome 11.

A Breast Cancer-Associated SNP Adjacent to ESR1 Correlates with Oestrogen Receptor-α (ERα) Level in Invasive Breast Tumours.

Abstract Aim: To identify whether breast cancer-associated SNP rs2046210 is associated with ERα level in invasive breast tumours.BackgroundA recent genome-wide association study identified SNP rs2046210 at 6q25.1 as having a strong association with breast cancer risk1. The SNP is located 29kb upstream of the first untranslated exon of ESR1 and 180kb upstream of the transcription start site2. Rs2046210 is not in linkage disequilibrium (LD) with two of the most widely studied polymorphisms in ESR1. We test here the hypothesis that rs2046210 may be associated with altered ESR1 expression.MethodsPatients with both leukocyte DNA and invasive breast tumour paraffin blocks available were identified from two of our ongoing tissue collections: Femara Anastrazole Clinical Evaluation (FACE) and the British Breast Cancer (BBC) study. Germline DNA was extracted from bloods using the QIAamp DNA Blood Mini Kit. PCR was performed with primers spanning the SNP site, with a single basepair mismatch in the reverse sequence to generate an HhaI restriction site in the presence of the C allele. PCR product was run on a 3% agarose gel to confirm presence of a single 120bp band. Restriction digest with HhaI was then performed and products run on a 4% Metaphor agarose gel. Genotype was assigned as follows: 97bp band only = wild-type (C/C); 97bp and 120bp band = heterozygote (C/T); 120bp band only = variant (T/T). From corresponding invasive breast tumour, ERα was assessed on 4μm whole sections using clone 6F11 (Vector Labs) and quantified by H-score. Mean H-scores (left and right invasive breast tumours) were used for the BBC group. ERα- samples (H-score≤1.0) were excluded. Analysis was by ANOVA using non-parametric bias-corrected and accelerated 95% bootstrap confidence intervals (2000 replications), with genotype fitted as a score and study as a stratifying co-variate.ResultsH-score (mean) by genotypeStudyWild-typeHeterozygoteVariantFACE (n=280)171.8180.9184.9BBC (n=46)180.0178.9194.1Fitted mean172.6179.7186.7(% samples)(45)(44)(11) Minor allele frequency was 33%, which is similar to the 38% previously reported in patients of European ancestry1. Within FACE, increased ERα was seen with presence of variant SNP. In the BBC group, variant SNP patients had higher ERα than both wild-type and heterozygote. Overall, there was a significant difference in ERα score per genotype group of 7.05, (95% CI 0.7-13.5, p=0.035). This was circa 4% difference in H-score per variant allele.ConclusionThe variant genotype of SNP rs2046210 is associated with increased ERα expression. While the increase contributed by the variant allele is relatively modest, this may partly explain why the SNP is associated with increased breast cancer risk. Similar studies should be conducted in normal breast tissue.1Zheng et al. (2009) Nat. Gen. 41(3): 324-3282Kos et al. (2001) Mol. Endocrinol. 15: 2057-2063 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4138.

Establishment of a Highly Sensitive Allele Specific Wild Type Blocker Polymerase Chain Reaction Followed by Pyrosequencing for Detection of the p.D816V Point Mutation.

Abstract 4975 BackgroundThe vast majority of mastocytosis patients carry the p.D816V activating point mutation in exon 17 leading to imatinib resistance. In addition, several less common activating kit mutations, at least partially accessible to imatinib, have been described in a minority of systemic and cutaneous mastocytosis. Therefore, exact molecular identification of patients with mastocytosis of uncertain dignitiy is a precondition for diagnostic and therapeutic purposes. However, atypical mast cell infiltrates within the bone marrow biopsies often are very small and microdissection may be hampered by the inconspicuousness of the atypical mast cells. Hence, an appropriate molecular test should exhibit an exceedingly high sensitivity. MethodsIn an attempt to combine high sensitivity and robustness we created an approach where we adapted the principle of wild-type blocker PCR employing LNA-substituted oligonucleotides and pyrosequencing technique. First, the unique properties of LNA substituted oligonucleotides were employed. As a consequence of their 2′-O, 4′-C methylene bridge and their characteristic bicyclic structure binding affinity is significantly increased, resulting in high melting points. One base mismatch between an LNA oligonucleotide and the complementary strand can lower the melting point 20-30°C, thereby allowing a LNA oligonucleotide to discriminate a one base pair difference between templates and, in contrary, a single base pair mismatch in the normal DNA octomer decreases Tm only by 10°C. In order to allow LNA-substituted oligonucleotides to block amplification of wild-type a mutated form of Taq polymerase, termed the Stoffel fragment was applied. Stoffel fragment is a modified form of AmpliTaq® DNA polymerase lacking intrinsic 5' to 3' exonuclease activity.Employing this methodological setting including the Stoffel fragment, the LNA oligonucleotides and appropriate primer 20 rounds of PCR were performed and an amplicon of 185 bp was received. As demonstrated employing mixtures of assembled DNA sequences keeping the WT or the p.D816V point mutation the WT/D816V ratio turned in favour of the mutated region performing the LNA – Stoffel PCR. Unique in our approach was a second step in which the suchlike amplified PCR product then was adopted in a pyrosequencing assay (Pyromark 24, Qiagen, Hilden, Germany). ResultsFirst of all, employing WT DNA sequences and the L1236 cell line, the ability of the blocking LNA oligonucleotides to prevent primer extension on WT-DNA by AmpliTaq® DNA polymerase was excluded. To determine the minimum concentration of blocker necessary to prevent amplification of WT oligonucleotides, we used serial dilutions of the blocking oligonucleotides and revealed that a minimum of 50 mM of blocker was necessary to prevent a highly competitive WT-amplicon. We then analyzed the sensitivity of the assay and spiced assembled DNA sequences keeping the WT or the D816V point mutation. Performing this experiment, 1:105 (0,001%) mutant DNA sequences were definitely detectable performing this assay but not performing conventional pyrosequencing without specific preamplification. In none of the control experiments employing WT DNA a false positive amplicon of the exon 16 D816V region was seen indicating an excellent specifity of the assay. In the pyrograms only light emission peaks >10% were evaluated.To determine the utility of our technique for detecting minority mutations in bone marrow specimen containing few cells or mixed cell populations, we performed the here presented assay on genomic DNA extracted from formalin-fixed, EDTA decalcified paraffin-embedded trephine biopsies. In all 20 trephine biopsies under study, comprising a morphologically established atypical mast cell density from 1 – 15% a clear cut decision of the genomic status was possible. ConclusionsWild-type blocker PCR employing nonextendable LNA oligonucleotides represents an exceedingly sensitive, allele-specific method. In combination with the pyrosequencing technique the here presented assay is highly reliable and reproducible, simple and fast to perform (3 h), facile to interpret and should be equally applicable to other single-base mutations. In bone marrow biopsies mRNA fragments often are highly degradated. Therefore, in contrast to PCR assays performed on cDNA, the here presented assay is ideal suited for the molecular diagnosis of systemic mastocytosis on formalin-fixed tissues. DisclosuresSiebolts:Novartis: Research Funding. Al-Ali:Novartis:. Niederwieser:Novartis: Research Funding. Wickenhauser:Novartis: Research Funding.

Effect of Single Central Base Pair Mismatch on the Conformation and Stability of Surface Immobilized DNA: Molecular Dynamics Studies

We combine molecular dynamics simulations and relative free energy calculation to investigate how single central base mismatch influence the conformation and stability of surface immobilized DNA. For comparison purpose, molecular dynamics simulations have been performed on a central mismatched duplex and the corresponding perfectly matched duplex, respectively. Simulations have been conducted with the generalized Born model. The relative free energy differences of the two systems suggest the single base-pair mismatch may lower the stability of DNA as expectation. The result obtained from the structural analysis shows the average conformations of the two DNA duplexes resemble the B-form, which suggests the central base pair mismatch has little effect on the overall conformation of DNA. Furthermore, our result shows the fluctuation of the perfectly matched duplex is obviously larger than that of the central base pair mismatched duplex. This study gives us a new atomic-level insight into DNA probe-target interaction on the surface. The results presented here will also aid in the design of DNA microarrays for single nucleotide polymorphisms screening.

Assessment of Primer/Template Mismatch Effects on Real-Time PCR Amplification of Target Taxa for GMO Quantification

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

Enhancing allele-specific PCR for specifically detecting short deletion and insertion DNA mutations

Allele-specific PCR (AS-PCR) has been widely used for the detection of single nucleotide polymorphism. But there are some challenges in using AS-PCR for specifically detecting DNA variations with short deletions or insertions. The challenges are associated with designing selective allele-specific primers as well as the specificity of AS-PCR in distinguishing some types of single base-pair mismatches. In order to address such problems and enhance the applicability of AS-PCR, a general primer design method was developed to create a multiple base-pair mismatch between the primer 3′-terminus and the template DNA. This approach can destabilize the primer–template complex more efficiently than does a single base-pair mismatch, and can dramatically increase the specificity of AS-PCR. As a proof-of-principle demonstration, the method of primer design was applied in colony PCR for identifying plasmid DNA deletion or insertion mutants after site-directed mutagenesis. As anticipated, multiple base-pair mismatches achieved much more specific PCR amplification than single base-pair mismatches. Therefore, with the proposed primer design method, the detection of short nucleotide deletion and insertion mutations becomes simple, accurate and more reliable.

Sequence‐Specific HCV RNA Quantification Using the Size‐Dependent Nonlinear Optical Properties of Gold Nanoparticles

The hepatitis C virus (HCV) is a single-stranded (ss) RNA virus that is responsible for chronic liver diseases, such as cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Driven by the need to detect the presence of the HCV viral sequence, herein it is demonstrated for the first time that the nonlinear optical (NLO) properties of gold nanoparticles can be used for screening and quantifying HCV RNA without any modification, with excellent detection limit (80 pM) and selectivity (single base-pair mismatch). The hyper-Rayleigh scattering (HRS) intensity increases 25 times when label-free, 145-mer, HCV ss-RNA is hybridized with 400 pM target RNA. The mechanism of HRS intensity change is discussed with experimental evidence for a higher multipolar contribution to the NLO response of gold nanoparticles.

Macrosolute Effects on Nucleic Acid Interactions

The intracellular environment contains a variety of solutes that cumulatively occupy a significant volume of the cell (20-30%). The high volume occupancy generates a system which is macromolecularly crowded. This crowding, also known as the excluded volume effect, can lead to an increase in the chemical activity of solutes and influence thermodynamic and kinetic values as compared to a dilute system. Using synthetic, inert cosolutes to provide a simplified mimic of the crowding in the intracellular environment, DNA structures were studied. We demonstrate that crowding can lead to the differential stabilization of a complementary DNA duplex over duplexes containing a single mismatched base pair, effectively increasing the specificity of the hybridization reaction. In systems with molecularities ranging from one to four we demonstrate that as the molecularity of a system increases the crowding effects also increase. An increase in Tm of up to 12°C was noted for a multi-branch DNA structure with four arms. Crowding mediated enhancement of the rate of hybridization was found to be independent of sequence but dependent upon structure.

A Label-Free, Force-Based Microarray Sensor

Within the past 15 years, force spectroscopy on single molecules has evolved from first proof-of-principles to a sophisticated method for the investigation of mechanics, folding kinetics, and complex formation of biomolecules. The quantification and characterization of molecular interactions is key to pharmaceutical and medical research, since it facilitates the development of therapeutics and serves as the basis of many diagnostic techniques. Force-based ligand detection offers many advantages over conventional approaches based on the thermodynamics of the interaction. It is label-free and permits crowded ambients. However, it requires very expensive equipment as well as labor intensive and time-consuming protocols. More severely, since force spectroscopy is inherently limited by thermal fluctuations, a molecular complex under investigation has to be probed thousands of times in order to achieve sufficient force resolution. This confines its application to low throughput formats. Here we present a high-throughput force spectroscopy approach in a parallel format, which in addition allows the detection of subtle changes in mechanical stability below the ones e.g. caused by a single base-pair mismatch in dsDNA. A very low affinity ATP selective DNA aptamer was implemented into a microarray compatible differential force detector design, wherein the relative stability of an aptamer·ligand complex is probed against a constant dsDNA reference complex. We found that the label-free assay selectively quantifies the concentration of ATP and that it reliably operates in a challenging, molecular crowded environment. The simplicity of the assay qualifies it as a tool that can be used in any laboratory equipped with basic fluorescence microscopy.

Mechanism of DNA Strand Exchange at Liposome Surfaces Investigated Using Mismatched DNA

DNA strand exchange is of great importance in vivo for genetic recombination and DNA repair. The detailed mechanism of strand exchange is not understood in full detail despite extensive studies. Simplistic model systems in which molecular parameters can be varied independently are therefore of interest to study. We chose the surface of a positively charged liposome as a scaffold, which we recently demonstrated to be able to catalyze the exchange of fully complementary DNA oligonucleotides. We here study how single base pair mismatches affect the rate of strand exchange on the liposome surface. Interestingly, the rate of the exchange does not simply follow the stability of the duplex in solution, as determined by melting temperatures, but also depends sensitively on the position of the mismatch. For duplexes with similar melting temperatures, the exchange is much faster for a mismatch close to the end than for a mismatch in the middle of the sequence. Our results suggest that the single strands are stabilized by the liposome surface; therefore, the duplex is fraying more and the DNA opens up in a zipperlike fashion on the surface, increasing the probability of strand exchange. We also show that the competition between greater stability (higher Tm in solution) and higher concentration is important for the final composition of the duplex when a large excess of single strands is added to a complementary double-stranded DNA. Finally, the similar exchange rate constants for fully base-paired duplexes on the liposome surface when adding fully matched single strands or single strands with a mismatched base indicate that the rate is governed largely by separation of the initial duplex and not by the formation of the product duplex.

Single Base Pair Mutation Detection by PNA Direct PCR Clamping: Application to Bcr-Abl T315I Mutation

Background: Mutations occurring within the kinase domain of the tyrosine kinase c-Abl, and its oncogenic counterpart Bcr-Abl, strongly affects the outcome of clinical treatment with tyrosine kinase inhibitors (TKIs). Although a second and third generation of TKIs has been developed and these molecules are currently available some mutations (i.e. T315I) confer insensitivity to the drugs. Therefore, a method allowing quick detection of these mutations still represents a challenge for most of clinicians because it would allow them to rapidly switch to alternative therapies instead of TKIs. Preliminary data of a novel method allowing easy and rapid detection of the mutation affecting the codon 315 (T315I), which is nowadays the most severe and resistant mutation to TKIs, is presented.Rationale & Methods: The innovative strategy we propose is based on a variant of a single tube PNA-PCR clamping. Peptide Nucleic Acid (PNA) are synthetic molecules sharing physical-chemical features with DNA. Indeed, they possesses a structural backbone formed by N-(2-amino-ethyl)-glycine, instead of deoxy-ribose molecules linked together by phosphodiester bond, covalently conjugated with the four bases (A, T, G, C). This peculiar structure confers to the molecule exceptionally high stability when matches a single complementary DNA strand. Interestingly, the binding of PNA to nucleic acids is not affected by ionic strength and, compared to identical homo-duplex DNA-DNA, displays higher melting temperature (Tm). The latter feature allows detection of point mutations because a single mismatch within the sequences would lower the Tm of approximately 20°C. Taking advantage of these unique PNA properties, together with the fact that PNA can not prime DNA polymerase job, we utilized sequence specific PNA to block PCR amplification. The approach makes use of a specific clamping mechanism, the “primer exclusion”, in which PNA and oligonucleotide primer compete for the same target sequence. As consequence when PNA binds to template DNA the DNA polymerase can not synthesize new DNA strand. The experimental design we propose schedules the use of a PNA harboring a sequence which perfectly matches within the wild-type (wt) c-Abl tyrosine kinase domain. Therefore, when a single base pair mismatch occurs, such as it happens in presence of mutation conferring TKIs resistence, the hetero-duplex PNA-DNA stability is strongly impaired. The expected PCR clamping readout would be as follow:in case of wt template PCR reaction will not work because DNA polymerase activity is impaired by the formation of PNA-DNA hetero-duplex; on the other hand whena mutation occurs the stability between PNA and DNA complementary sequence is compromised allowing oligonucleotide primer to displace PNA.So far we focused our efforts on the mutation affecting codon 315 (T315I).Results & Conclusions: cDNA synthesized from total RNA isolated from patients harboring either wt or mutated Bcr-Abl alleles have been amplified by nested PCR. During the first step a specific pair of primers designed to discriminate between the kinase domain of c-Abl and Bcr-Abl have been utilized. The second step involves the use of sequence specific 19-mer PNA matching within the region around the codon 315 (T315I), so far the most severe mutation described, in a “PNA primer exclusion” PCR clamping reaction. Initially, we aimed to study patients which have previously been analyzed by direct sequencing in which either the wt or the mutated allele were to be predominant. Encouraging results show amplification in patients harboring the point mutation at position 315 which determine the substitution of a threonine for isoleucine (T315I). These results were further corroborated by carrying-out a blind analysis, in which samples were subsequently sequenced. These data show that, in future, the technique herein reported might be extremely useful in detecting the presence of all known mutations within the Bcr-Abl kinase domain with simple, rapid, reliable and very cheap methodology.

Electrostatic readout of DNA microarrays with charged microspheres.

DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-mum lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.

Gold‐Nanorod‐Based Sensing of Sequence Specific HIV‐1 Virus DNA by Using Hyper‐Rayleigh Scattering Spectroscopy

Infectious diseases caused by the human immunodeficiency virus (HIV) remain the leading killers of human beings worldwide, and function to destabilize societies in Africa, Asia, and the Middle East. Driven by the need to detect the presence of HIV viral sequence, here we demonstrate that the second-order nonlinear optical (NLO) properties of gold nanorods can be used for screening HIV-1 viral DNA sequence without any modification, with good sensitivity (100 pico-molar) and selectivity (single base-pair mismatch). The hyper-Rayleigh scattering (HRS) intensity increases 45 times when a label-free 145-mer, ss-gag gene DNA, was hybridized with 100 pM target DNA. The mechanism of HRS intensity change has been discussed with experimental evidence for higher multipolar contribution to the NLO response of gold nanorods.

A label-free DNA sensor based on impedance spectroscopy

This paper describes a label-free detection system for DNA strands based on gold electrodes and impedance measurements. A single-stranded 18 mer oligonucleotide (ssDNA) was immobilised via a thiol linker on gold film electrodes and served as probe DNA. Residual binding places were filled with mercaptobutanol. The sensor surface clearly distinguished between complementary and non-complementary target ssDNA. Additionally, detection of single base pair mismatches was possible. The electrode was impedimetrically characterised in the presence of the redox system ferri/ferrocyanide before and after DNA hybridisation. Impedance analysis showed that the charge transfer resistance, R ct, was increasing after DNA duplex formation, whereas the capacitive properties remain rather unaltered. The relative change of R ct was used as sensor parameter. Concentrations in the nanomolar range have been detected by the system. The sensor was reusable because a denaturation protocol allowed effective double strand dissociation without changing the surface properties of the electrode substantially. The time for DNA detection have been reduced to about 15 min including regeneration. The sensor signal was amplified by about 20% after binding of a negatively charged molecule to the formed DNA duplex. The sensor was also capable of sensing longer target ssDNA strands as shown with 25 mer and 37 mer oligonucleotides.

Electrochemical Detection of Short DNA Sequences Related to the Escherichia coli Pathogen Using a Zirconia-Modified Screen-Printed DNA Biosensor

A simple, disposable and inexpensive electrochemical DNA biosensor based on a zirconia (ZrO2) modified thin film screen-printed electrode (ZrO2/SPE) has been developed. Short DNA sequences (21 monomer units) from the Escherichia coli pathogen, modified with a phosphate group at the 5′ end, were attached to the surface of the electrode through the affinity of the phosphate group for zirconia, to produce an effective DNA probe (ssDNA/ZrO2/SPE). DNA immobilization and hybridization were characterized using differential pulse voltammetry by employing methylene blue as redox indicator. Target sequences hybridized with the probe resulted in a decrease of the reduction peak current of methylene blue intercalated into the probe. The response of a non-complementary sequence and a single base pair mismatch sequence were both clearly distinguished from that of a complementary sequence. The developed biosensor had a high selectivity and sensitivity towards hybridization detection (10–10 M complementary DNA detectable). Making use of screen-printed technology, the fabrication of the biosensors exhibited satisfactory reproducibility, investigated by cyclic voltammetry and differential pulse voltammetry. The relative standard deviation was found to be <3.0% for six bare SPEs and six ssDNA-modified SPEs (ssDNA/ZrO2/SPE) from a batch.

Selective oxidation of methylcytosine in single base pair mismatches by osmium complex

We have reported a novel method for methylcytosine (M)-positive epigenotyping, in which an osmium ion site-specifically binds to M in a single strand or a bulge structure of DNA on oxidation of an M C5-C6 double bond. Herein, we have examined the osmium oxidation reactivity of M that formed a mismatched base pair with various bases, and observed its unique reactivity.

Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10(8) bound targets per cm(2) sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.