Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis.Based on the hyperbranched rolling circle amplification(HRCA) and microarray techniques,a new method for qualitative detection of methylation was developed.In the present study,padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes.Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction.Utilizing the method of 3-dimensional polyacrylamide gel-based microarray,the HRCA product will be immobilized on the slide to form a DNA microarray,which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.To control the false positive signals,DNA ligase and temperature of ligation/digestion are optimized.Methylation status of four CpG sites located in P15,Ecadherin,hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR.Our research proves that this method is simple and inexpensive,and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.     

Signal amplification by rolling circle amplification on universal flaps yielded from target-specific invasive reaction

Detection of nucleic acids with signal amplification is preferable in clinical diagnosis. A novel approach was developed for signal amplification by coupling invasive reaction with hyperbranched rolling circle amplification (HRCA). Invasive reaction, which does not rely on specific recognition sequences in a target but a specific structure formed by the specific binding of an upstream probe and a downstream probe to a target DNA, can generate thousands of flaps from one target DNA; then the flaps are ligated with padlock probes to form circles, which are the templates of HRCA. As HRCA amplicon sequence is free of target DNA sequence, signal amplification is achieved. Because flap sequence is the same to any target of interest, HRCA is universal; the detection cost is hence greatly reduced. The sensitivity of the proposed method is less than 1 fM artificial DNA targets; and the specificity of the method is high enough to discriminate one base difference in the target sequence. The feasibility was verified by detecting real biological samples from HBV carriers, indicating that the method is highly sensitive, cost-effective, and has a low risk of cross-contamination from amplicons. These properties should give great potential in clinical diagnosis.     

Non‐hybridization single nucleotide polymorphism detection and genotyping assay through direct discrimination of single base mutation by capillary electrophoretic separation of single‐stranded DNA

The significant demands for single nucleotide polymorphism detection and genotyping assays have grown. Most common assays are based on the recognition of the target sequence by the hybridization with its specific probe having the complementary sequence of the target. Herein, a simple, label‐free, and economical non‐hybridization assay was developed for single nucleotide polymorphism detection and genotyping, based on the direct discrimination of single base mutation by simple capillary electrophoresis separation for single‐stranded DNA in an acidic electrophoretic buffer solution containing urea. Capillary electrophoresis separation of single‐base sequential isomers of DNA was achieved due to charge differences resulting from the different protonation properties of the DNA bases. Single nucleotide polymorphism detection and genotyping were achieved by discriminating the electropherogram pattern change, that is, peak number in the electropherogram, obtained by the proposed method. The successful practical application of the proposed method was demonstrated through single nucleotide polymorphism detection and genotyping on a known gene region of 84‐mer, in which guanine to adenine single‐base mutation is commonly observed, using a human hair sample in combination with genomic DNA extraction, polymerase chain reaction amplification, DNA purification from polymerase chain reaction products, and capillary electrophoresis separation.     

Chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms on a solid surface using target-primed rolling circle amplification

A new chemiluminescent (CL) method has been developed for the sensitive detection of DNA hybridization and single-nucleotide polymorphisms (SNPs) with target-primed rolling circle amplification (RCA). The capture oligonucleotide probe is firstly immobilized on a polystyrene well plate and then hybridized with the wild DNA target. A designed padlock probe is circularized after perfect hybridization to the DNA target. Then the RCA reaction can be initiated from the DNA target that acts as a primer and generates a long tandem single-strand of DNA with repeat sequences. In contrast, the mutant DNA target, which contains a mismatched base with the padlock probe, cannot initiate the RCA reaction and primes only a limited extension with the unligated padlock probe. Afterwards, a biotinylated oligonucleotide is used to hybridize with the RCA product in each repeat sequence and streptavidin-alkaline phosphatase (STV-AP) is employed to combine the anchored biotin. The DNA target is detected with the CL reaction of STV-AP and 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). With the RCA-based method, the sensitivity of DNA detection can be increased by about two orders of magnitude compared with that of direct DNA hybridization. A DNA target as low as 3.6 pM can be detected. Wild-type DNA and the one-base mutant DNA can be differentiated with high selectivity through this RCA reaction.     

Structure-specific DNA cleavage on surfaces

The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.     

Hairpin Assembly Amplified Electrochemical Biosensor for Highly Sensitive and Specific Detection of DNA

In this report, a simple electrochemical biosensor has been developed for highly sensitive and specific detection of DNA based on hairpin assembly amplification. In the presence of target DNA, the biotin‐labelled hairpin H1 is opened by hybridizing with target DNA through complementary sequences. Then the opened hairpin H1 assembles with the hairpin H2 to displace the target DNA, generating H1‐H2 complex. The displaced target DNA could trigger the next cycle of hairpins assembly, resulting in the generation of numerous H1‐H2 complexes. Subsequently, the H1‐H2 complex hybridizes with the capture probe immobilized on the electrode. Finally, the streptavidin alkaline phosphatase (ST‐ALP) binds to biotin in the capture probe‐H1‐H2 complex and catalyzes the substrate α‐naphthol (α‐NP) to produce electrochemical signal. To make a more fascinating hairpin assembly amplification strategy in signal amplification, mismatched base sequences are designed in hairpin H2 to decrease non‐specific binding of the hairpin substrates. The developed biosensor achieves a sensitivity of 20 pM with a linear range from 25 pM to 25 nM, and shows high selectivity toward single‐base mismatch. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.     

Electronic transduction of DNA sensing processes on surfaces: amplification of DNA detection and analysis of single-base mismatches by tagged liposomes.

Tagged, negatively charged, liposomes are used to amplify DNA sensing processes. The analyses of the target DNA are transduced electrochemically by using Faradaic impedance spectroscopy, or by microgravimetric measurements with Au-quartz crystals. By one method, a probe oligonucleotide (1) is assembled on Au-electrodes or Au-quartz crystals. The formation of the double-stranded assembly with the analyte DNA (2) is amplified by the association of the 3-oligonucleotide-functionalized liposomes to the sensing interface. The target DNA is analyzed by this method with a sensitivity limit that corresponds to 1 x 10(-12) M. A second method to amplify the sensing of the analyte involves the interaction of the 1-functionalized electrode or Au-quartz crystal with the target DNA sample (2) that is pretreated with the biotinylated oligonucleotide (4). The formation of the three-component double-stranded assembly between 1/2/4 is amplified by the association of avidin and biotin-labeled liposomes to the sensing interfaces. By the secondary association of avidin and biotin-tagged liposomes, a dendritic-type amplification of the analysis of the DNA is accomplished. The analyte DNA (2) is sensed by this method with a sensitivity limit corresponding to 1 x 10(-13) M. The biotin-tagged liposomes are also used to probe and amplify single-base mismatches in an analyte DNA. The 6-oligonucleotide-functionalized Au-electrode or Au-quartz crystal was used to differentiate the single-base mismatch (G) in the mutant (5) from the normal A-containing gene (5a). Polymerase-induced coupling of the biotinylated-C-base to the double-stranded assembly generated between 6 and 5 followed by the association of avidin and biotin-tagged liposomes is used to probe the single base mismatch. The functionalized liposomes provide a particulate building unit for the dendritic amplification of DNA sensing.     

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.     

Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification （RCA） technique. In the presence of DNA adenine methylation （Dam） MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons （detection probes）, resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.     

DNA analysis by application of Pt nanoparticle electrochemical amplification with single label response

This study demonstrates a highly sensitive sensing scheme for the detection of low concentrations of DNA, in principle down to the single biomolecule level. The previously developed technique of electrochemical current amplification for detection of single nanoparticle (NP) collisions at an ultramicroelectrode (UME) has been employed to determine DNA. The Pt NP/Au UME/hydrazine oxidation reaction was employed, and individual NP collision events were monitored. The Pt NP was modified with a 20-base oligonucleotide with a C6 spacer thiol (detection probe), and the Au UME was modified with a 16-base oligonucleotide with a C6 spacer thiol (capture probe). The presence of a target oligonucleotide (31 base) that hybridized with both capture and detection probes brought a Pt NP on the electrode surface, where the resulting electrochemical oxidation of hydrazine resulted in a current response.     

Development of a lipase-based optical assay for detection of DNA

A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate a detectable readout the captured lipase was applied to an optical assay that takes advantage of the enzymatic activity of lipase. The assay applies p-nitrophenol octanoate (NPO) as the substrate and in the presence of lipase the ester is hydrolyzed to p-nitrophenolate which has a strong absorbance at 405 nm. The method provides detection a detection limit of 200 fmol target DNA and it was able to distinguish single base mismatches from the fully complementary target.     

Simultaneous electrochemical determination of two analytes based on nuclease-assisted target recycling amplification

In the present study, a method for simultaneous determination of two different DNAs is developed based on nuclease-assisted target recycling and nanoparticle amplification. The target recycling process is accomplished by taking advantage of the cleavage property of nicking endonuclease (NEase) for specific nucleotide sequences in duplex. In the presence of target DNA, the linker DNA in our detection system can hybridize with the target and be cleaved to form short fragments. Thus the target DNA is released and recognized by another linker DNA, activating the next round of cleavage reaction. On the other hand, two bio-barcode probes, a PbS nanoparticles (NPs)-DNA probe and a CdS NPs-DNA probe, are used for tracing two target DNAs to further amplify the detection signals. Based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Pb²⁺ and Cd²⁺ obtained by dissolving two probes, two different target DNAs are determined with high sensitivity and single-base mismatch selectivity.     

Multiplexed Immunosensing Platform Coupled to Hybridization Chain Reaction for Electrochemical Determination of MicroRNAs in Clinical Samples

There is an urgent need for development of rapid and inexpensive techniques for detection of microRNAs (miRNAs), which are potential biomarkers of various types of cancer. In this paper, we describe a multiplexed electrochemical platform for determination of three cancer‐relevant miRNAs: miR‐21, let‐7a and miR‐31. The strategy combines the use of magnetic beads (MBs) modified with a commercial antibody for the efficient capture of the heteroduplexes formed by hybridization of the target miRNA with DNA probe. Free non‐hybridized region of the DNA probe was thereafter hybridized with two biotin‐labeled auxiliary DNA probes in a process of hybridization chain reaction (HCR), resulting in a long hybrid bearing a large number of biotin molecules. Labeling of these multiple biotin units with streptavidin‐peroxidase conjugates allowed an amplification of the amperometric signal measured after capturing the modified MBs at a screen‐printed carbon electrode array of eight electrodes. The combined strategy demonstrated in a similar assay time significantly higher sensitivity than those previously described using modified MBs with the same capture antibody (without amplification by HCR) or a HCR strategy implemented on the surface of MBs, respectively. The methodology exhibits a good selectivity for discriminating single mismatches and was applied to the determination of the three target miRNAs in total RNA (RNA_t) extracted from various cancer cell lines and from cervical precancerous lesions.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

Ultrasensitive fluorescence polarization DNA detection by target assisted exonuclease III-catalyzed signal amplification

Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.     

一种基于微芯片电泳平台的级联化学发光信号放大策略用于HIV-DNA的高灵敏检测

该文基于微芯片电泳-化学发光检测（MCE-CL）平台,以辣根过氧化酶标记的DNA(HRP-DNA)作为信号探针,利用HRP 催化鲁米诺和双氧水化学发光反应及目标分子与DNA的杂交反应,结合T7Exo酶辅助信号放大,建立了一种MCE分离辅助双循环化学发光信号放大的新方法。结果显示：优化实验条件下,在1.0×10-14~5.0×10-9 mol/L范围内,HIV-DNA的浓度对数值与HIV-DNA的化学发光强度呈良好的线性关系,检出限（S/N=3）为1.6×10-15 mol/L,在0.10、0.25、1.0、10（×10-12 mol/L）4个加标水平下的回收率为93.0%～103%,相对标准偏差（RSD）为0.50%～3.7%,方法具有较好的准确度,可应用于人血清中HIV-DNA的高灵敏检测。     

Human Y‐chromosome SNP characterization by multiplex amplified product‐length polymorphism analysis

We designed an allele‐specific amplification protocol to optimize Y‐chromosome SNP typing, which is an unavoidable step for defining the phylogenetic status of paternal lineages. It allows the simultaneous highly specific definition of up to six mutations in a single reaction by amplification fragment length polymorphism (AFLP) without the need of specialized equipment, at a considerably lower cost than that based on single‐base primer extension (SNaPshot?) technology or PCR‐RFLP systems, requiring as little as 0.5 ng DNA and compatible with the small fragments characteristic of low‐quality DNA. By designation of two primers recognizing the derived and ancestral state for each SNP, which can be differentiated by size by the addition of a noncomplementary nucleotide tail, we could define major Y clades E, F, K, R, Q, and subhaplogroups R1, R1a, R1b, R1b1b, R1b1c, J1, J2, G1, G2, I1, Q1a3, and Q1a3a1 through amplification fragments that ranged between 60 and 158bp.     

Characterization of tectoRNA assembly with cationic conjugated polymers

Association between RNAs with preprogrammed molecular recognition units can be quantified by using cationic, water-soluble conjugated polymers. The method uses a fluorophore-labeled probe RNA (RNA-F*), which is treated with a target structure (RNA-T). Heterodimer formation, (RNA-T/RNA-F*), increases the total negative charge on the F*-bearing macromolecule and reduces the number of negatively charged molecules (relative to unbound RNA-T+ RNA-F*). On the basis of electrostatic interactions, we anticipated more effective binding between CCP and (RNAT/RNA-F*), a reduction of the average CCP- - -F* distance, and more effective FRET upon excitation of the conjugated polymer. The resulting signals benefit from the optical amplification characteristic of emissive conjugated polymers. Solution dissociation constants can be determined by monitoring F* intensity changes as a function of [RNA-F*] and the ratio: [I(T) - I(NB)]/I(NB), where I(T) and I(NB) are the F* intensities in the presence of the target RNA (RNA-T) and a nonbinding RNA (RNA-NB), respectively, while keeping the concentration of the conjugated polymer constant. By focusing on [I(T) - I(NB)]/I(NB) as a function of RNA concentration, one can detect the concentration range wherein increased fluorescence is the result of dimerization.     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.