一种DNA扩增的新技术： 利用热稳定的Bst DNA聚合酶驱动跨越式滚环等温扩增反应

Bst DNA聚合酶具有热稳定性、链置换活性及聚合酶活性,在体外DNA等温扩增反应中起重要作用. 本文利用Bst DNA聚合酶的5′→3′聚合酶、核苷酸（末端）转移酶及链置换酶活性发展了一种新的体外环式DNA扩增技术跨越式滚环等温扩增(saltatory rolling circle amplification,SRCA)．在SRCA反应中,Bst DNA聚合酶以上游引物P1为模板合成其互补链RcP1,并和P1形成双链DNA|之后,Bst DNA聚合酶用其核苷酸转移酶活性在其P1的3′末端沿5′→3′方向随机掺入脱氧核糖核苷酸聚合形成寡聚核苷酸（dNMP)m序列,即DNA的合成反应跨越了RcP1 与下游引物P2之间的缺口|然后,以下游引物P2为模板形成互补序列（RcP2）;接着,Bst DNA聚合酶继续将脱氧核糖核苷酸随机添加到RcP2的3′末端,形成（dNMP)n序列．继而,Bst DNA聚合酶以RcP1为模板,继续催化聚合反应合成互补新链,并通过其链置换酶活性替换P1|如此往复,形成［P1-(dNMP)m-RcP2-(dNMP)n …］序列．本文通过电泳、酶切、测序等方法对扩增产物进行分析,演绎出上述扩增过程,并就工作原理进行了讨论．该反应可能对开发等温扩增技术检测微生物有一定助益,也为解释环介导等温扩增技术中假阳性反应和滚环等温扩增反应中的背景信号提供了线索．     

Enzymatic preparation of multimilligram amounts of pure single-stranded DNA samples for material and analytical sciences

We present a method for high-yield production of multimilligram amounts of pure single-stranded DNA employing rolling circle amplification (RCA) and processing by restriction enzymes. Pure and homogeneous samples are produced with minimal handling time, reagents, and waste products. The RCA method is more than twice as efficient in dNTP incorporation than conventional polymerase chain reaction in producing end product. The validity and utility of the method are demonstrated in the production of a uniformly ¹³C/¹⁵N-labeled 38-nt cocaine aptamer DNA used in nanosensing devices.     

Double-probe signal enhancing strategy for toxin aptasensing based on rolling circle amplification

On the basis of aptamer-based rolling circle amplification (RCA) and magnetic beads (MBs), a highly sensitive electrochemical method was developed for the determination of Ochratoxin A (OTA). Initially, an amino-modified capture DNA was immobilized onto MBs for the following hybridization with an OTA aptamer and a phosphate labeled padlock DNA. In the presence of OTA, the aptamer would dissociate from the bioconjugate, and the padlock DNA would subsequently hybridize with the capture DNA to form a circular template with the aid of the T4 ligase. Next, capture DNA would act as primer to initiate a linear RCA reaction and hence generate a long tandem repeated sequences by phi29 DNA polymerase and dNTPs. Then, two quantum dots (QDs) labeled DNA probes were tagged on the resulted RCA product to indicate the OTA recognition event by electrochemical readout. This strategy, based on the novel design of OTA-mediated DNA circularization, the combination of RCA and double signal probes introduction, could detect OTA down to the level of 0.2 pg mL(-1) with a dynamic range spanning more than 4 orders of magnitude. The proposed approach is tested to determine OTA in red wines and shows good application potential in real samples.     

Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

Background  

In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature.     

Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences

Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.     

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage–religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3 pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.     

Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology

Listeria monocytogenes (L. monocytogenes) is one of the most problematic human pathogens, as it is mainly transmitted through the food chain and cause listeriosis. Thus, specific and sensitive detection of L. monocytogenes is required to ensure food safety. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with magnetic beads based electrochemiluminescence (ECL) to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. At first, a linear padlock probe was designed to target a specific sequence in the hly gene which is specific to L. monocytogenes and then ligated by Taq DNA ligase. After ligation and digestion, further amplification by HRCA with a biotiny labeled primer and a tris (bipyridine) ruthenium (TBR) labeled primer was performed. The resulting HRCA products were then captured onto streptavidin-coated paramagnetic beads and were analyzed by magnetic beads based ECL platform to confirm the presence of targets. Through this approach, as low as 10 aM synthetic hly gene targets and about 0.0002 ng/μl of genomic DNA from L. monocytogenes can be detected, the ability to detect at such ultratrace levels could be attributed to the powerful amplification of HRCA and the high sensitivity of current magnetic bead based ECL detection platform.     

Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A

In this work, a new signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamer complementary (DNA2) were immobilized onto a magnetic bead (MB). In the presence of OTA, DNA2 was dissociated and released from the MB. The released DNA2 then hybridized with DNA3, which was linked at the 5' terminus of the amplification template and can extend along the template in the presence of Phi 29 DNA polymerase. The formed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI and produced a short single-stranded DNA. The cleaved DNA strand generated a new site by Phi 29 DNA polymerase and the process of extension and cleavage was cyclical. Thus, a amount of the short single-stranded DNA were produced. Using DNA and ABEI labeled carboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-stranded DNA could be sensitively detected. The CL intensity (ΔI) versus the concentration of OTA was linear in the range from 1.0×10(-12) to 5.0×10(-8)gmL(-1). The detection limit was 3.0×10(-13)gmL(-1), and the RSD was 3.4% at 1.0×10(-10)gmL(-1) (n=7). The developed method has been applied to detect OTA in naturally contaminated wheat samples. Due to its simplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CL bioassay offers a promising approach for the detection of OTA and other biomolecules.     

Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.     

Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.     

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

Matrix-assisted laser desorption/ionization mass spectrometry of immobilized duplex DNA probes.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry was used to analyze short DNA duplex probes with one strand immobilized on solid supports (straptavidin-coated magnetic beads or controlled pore glass beads). Only the non-immobilized strand could be detected. Partial denaturation was found when the duplex probes were mixed with 3-hydroxypicolinic acid, ammonium citrate matrix. The strategy has several applications, such as fast DNA sequence analysis and DNA diagnostics.     

TempliPhi,phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.     

A universal fluorescent aptasensor based on AccuBlue dye for the detection of pathogenic bacteria

We report a universal fluorescent aptasensor based on the AccuBlue dye, which is impermeant to cell membranes, for the detection of pathogenic bacteria. The sensor consists of AccuBlue, an aptamer strand, and its complementary strand (cDNA) that partially hybridizes to the aptamer strand. We have fabricated two models by changing the sequence of the reaction between the elements in the system. One is the “signal on” model in which the aptamer is first bound to the target, followed by the addition of cDNA and AccuBlue, at which time the cDNA hybridizes with the free unreacted aptamer and forms a double-stranded DNA (dsDNA) duplex. Such hybridization causes AccuBlue to insert into the dsDNA and exhibit significantly increased fluorescence intensity because of the specific intercalation of the AccuBlue into dsDNA rather than single-stranded DNA (ssDNA). The other model, “signal off,” involves hybridization of the aptamer with cDNA first, resulting in high fluorescence intensity on the addition of AccuBlue. When the target is added, the aptamer binds the target, causing the cDNA to detach from the dsDNA duplex and resulting in low fluorescence as a result of the liberation of AccuBlue. Because this design is based purely on DNA hybridization, and AccuBlue is impermeant to cell membranes, it could potentially be adapted to a wide variety of analytes.     

A fluorescent aptasensor for analysis of adenosine triphosphate based on aptamer–magnetic nanoparticles and its single‐stranded complementary DNA labeled carbon dots

A new fluorimetric aptasensor was designed for the determination of adenosine triphosphate (ATP) based on magnetic nanoparticles (MNPs) and carbon dots (CDs). In this analytical strategy, an ATP aptamer was conjugated on MNPs and a complementary strand of the aptamer (CS) was labeled with CDs. The aptamer and its CS were hybridized to form a double helical structure. The hybridized aptamers could be used for the specific recognition of ATP in a biological complex matrix using a strong magnetic field to remove the interfering effect. In the absence of ATP, no CDs–CS could be released into the solution and this resulted in a weak fluorescence signal. In the presence of ATP, the target binds to its aptamer and causes the dissociation of the double helical structure and liberation of the CS, such that a strong fluorescence signal was generated. The increased fluorescence signal was proportional to ATP concentration. The limit of detection was estimated to be 1.0 pmol L^–1 with a dynamic range of 3.0 pmol L^–1 to 5.0 nmol L^–1. The specific aptasensor was applied to detect ATP in human serum samples with satisfactory results. Moreover, molecular dynamic simulation (MDS) studies were used to analyze interactions of the ATP molecule with the aptamer.     

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R² = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.