Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)–short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n=11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.     

A microfluidic system for high-speed reproducible DNA sizing and quantitation

Microfabrication technology was used to develop a system consisting of disposable glass chips containing etched channels, reagents including polymer matrix and size standards, computer-controlled instrumentation for performing electrophoretic separations and fluorescence detection of double-stranded DNA, and software for automated data analysis. System performance was validated for separation and quantitation reproducibility using samples varying in amount and size of DNA fragments, buffer composition, and salt concentrations. Several applications of the microfluidic system for DNA analysis have been demonstrated, such as of polymerase chain reaction (PCR) products, sizing of plasmid digests, and detection of point mutations by restriction fragment length polymorphism (RFLP) mapping.     

Genotyping of single nucleotide polymorphism in MDM2 genes by universal fluorescence primer PCR and capillary electrophoresis

Single nucleotide polymorphism (SNP) 309 in the promoter region of the murine double minute 2 (MDM2) gene plays an important role in human tumorigenesis. We established a simple and effective CE method for SNP detection in the MDM2 gene. We designed one universal fluorescence-based nonhuman-sequence primer and one fragment-oriented primer, which were combined in one tube, and proceeded with the polymerase chain reaction (PCR). The amplicons were analyzed by capillary electrophoresis using single-strand conformation polymorphism method. PCR fragments generated from this two-in-one PCR displayed either T/T or G/G homozygosity or T/G heterozygosity. A total of 304 samples were blindly genotyped using this developed method, which included the DNA from 138 healthy volunteers, 43 chronic myeloid leukemia (CML) patients, and 123 colorectal cancer (CRC) patients. The results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE method was feasible for SNP screening of MDM2 in large populations.     

Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765 partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in such measurements. Figure Digital PCR amplification plot (left) and panel read out (right) of HindIII-digested pIRMM69. pIRMM69 contains one HindIII restriction enzyme site outside the hmg and transgene fragments used as targets in PCR. Red boxes with white shade denote positive hits containing one or more target DNA molecules, and white boxes with grey shade refer to no target being amplified.     

Quantitative Detection for Porphyromonas gingivalis in Tooth Pocket and Saliva by Portable Electrochemical DNA Sensor Linked with PCR

Here, a quantitative electrochemical analysis of periodontal bacteria in gingival crevicular fluid (GCF) and saliva by direct polymerase chain reaction (PCR) is presented. The electrochemical measurement was performed by mixing with PCR products and electrochemical indicator (bisbenzimidazole trihydrochloride). The peak current of indicator is reduced due to slower diffusion when the dye intercalates into the amplified DNA, and the degree of reduction in the peak current is correlates with the quantity of amplified DNA. Therefore, a quantitative analysis is possible by using our electrochemical method at the end point of PCR. In the GCF testing, The number of Porphyromonas gingivalis (Pg) detected by our electrochemical method at the end point of PCR were almost same compared with that were calculated by the conventional method of quantitative real? time PCR. In the saliva testing, the relationship between number of Pg in saliva and average pocket depth, and age‐dependence were also clearly observed. Since the saliva sample is obtained in a non‐invasive manner, this method is useful for the primary screening of periodontal disease. Moreover, our detection method is simple and uses a hand‐held potentiostat making it suitable for development of an on‐site periodontal diagnosis system.     

Principles of rapid polymerase chain reactions: mathematical modeling and experimental verification

Polymerase chain reaction (PCR) is an important diagnostic tool for the amplification of DNA. The PCR process can be treated as a problem in biochemical engineering. This study focuses on the development of a mathematical model of the polymerase chain reaction. The PCR process consists of three steps: denaturation of target DNA, annealing of sequence-specific oligonucleotide primers and the enzyme-catalyzed elongation of the annealed complex (primer:DNA:polymerase). The denaturation step separates the double strands of DNA; this model assumes denaturation is complete. The annealing step describes the formation of a primer-fragment complex followed by the attachment of the polymerase to form a ternary complex. This step is complicated by competitive annealing between primers and incomplete fragments including primer-primer reactions. The elongation step is modeled by a stochastic method. Species that compete during the elongation step are deoxynucleotide triphosphates dCTP, dATP, dTTP, dGTP, dUTP, and pyrophosphate. Thermal deamination of dCTP to form dUTP is included in the model. The probability for a species to arrive at the active site is based on its molar fraction. The number of random insertion events depends on the average processing speed of the polymerase and the elongation time of the simulation. The numerical stochastic experiment is repeated a sufficient number of times to construct a probability density distribution (PDF). The moment of the PDF and the annealing step products provide the product distribution at the end of the elongation step. The overall yield is compared to six experimental values of the yield. In all cases the comparisons are very good.     

Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.     

Continuous‐flow Polymerase Chain Reaction Microfluidics by Using Spiral Capillary Channel Embedded on Copper

Abstract

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene (PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous‐flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous‐flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s^?1, respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented‐flow PCR of three samples (249, 500 and 982 bp) has also been reported, which demonstrates the present continuous‐flow PCR microfluidics can be developed for high‐throughput genetic analysis.     

Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia.

This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1-17) and a short recombination distance from the low-density lipoprotein (LDL)-receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL-receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH-Savona and the D200G missense mutation in the exon 4, known as FH-Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody-protected polymerase were directly analyzed with an apparatus for high-voltage capillary electrophoresis on microchips and laser-induced fluorescence detection equipped with chips for the analysis of 25-500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from +0.60 to +/- 0.75 bp. This indicated that the size attribution to a conventional allele using the +/- 1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.     

Biochemical Properties and PCR Performance of a Family B DNA Polymerase from Hyperthermophilic Euryarchaeon <Emphasis Type="Italic">Thermococcus peptonophilus</Emphasis>

The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 °C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris–HCl (pH 8.0), 2 mM MgCl₂, 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 × 10⁻⁶) than Taq DNA polymerase (12.13 × 10⁻⁶) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time.     

Pyridyl‐Substituted Corrole Isomers: Synthesis and their Regulation to G‐quadruplex Structures

G‐quadruplex DNA plays an important role in the potential therapeutic target for the design and development of anticancer drugs. As various G‐quadruplex sequences in the promoter regions or telomeres can form different secondary structural modes and display a diversity of biology functions, variant G‐quadruplex interactive agents may be necessary to cure different disease by differentiating variant types of G‐quadruplexes. We synthesize five cationic methylpyridylium corroles and compare the interactions of corroles with different types of G‐quadruplexes such as cmyc, htelo, and bcl2 by using surface plasmon resonance. Because of the importance of human telomere G‐quadruplex DNA, we focus on the biological properties of the interactions between human telomere G‐quadruplex DNA and corrole isomers using CD, T_m, PCR‐stop (PCR= polymerase chain reaction), and polymerase‐stop assay, which demonstrate the excellent ability of the corrole to induce and stabilize the G‐quadruplex. This study provides the first experimental insight into how selectivity might be achieved for different G‐quadruplexes by a single group of methylpyridylium corrole isomers that may be optimized for potential selective cancer therapy.     

Kinetic studies exploring the role of anion templation in the slippage formation of rotaxane-like structures

The first examples of the slippage formation of rotaxane‐like structures in the presence of an anion template are reported between a macrocycle, synthesised by exploiting Eglinton coupling, and stoppered pyridinium axle components. The role of the anion template in the slippage process has been explored by kinetic studies. ¹H NMR spectroscopic investigations reveal the slippage species formed are not rotaxanes but pseudorotaxanes with some rotaxane character. The anion template significantly influences the amount of rotaxane character and the rate of slippage. Importantly, the fastest slippage rates, k_on, are achieved with the non‐coordinating hexafluorophosphate anion, whereas the slowest slippage off rates, k_off, are observed in the presence of coordinating anions, such as chloride. Since the k_off rates are significantly smaller than the k_on rates in the presence of coordinating anions, these anions act as templates favouring formation of the slippage species thermodynamically. Consequently, the resulting pseudorotaxanes with coordinating anions have greater rotaxane character. Two strategies for converting the slippage pseudorotaxanes into rotaxanes using hydrogenation or complexation with cobalt carbonyl are investigated.     

Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing‐based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost‐effective DNA length measurement for STRs and resequencing of small DNA fragments.     

Detection of Escherichia coli O157:H7 DNA using two fluorescence polarization methods

Using stx 2 gene in verotoxin-producing Escherichia coli O157:H7 as a target DNA, polymerase chain reaction (PCR) amplification has been combined with fluorescence polarization (FP) by two distinct combination protocols. The first approach (PCR-probe-FP) requires that fluorescence labeled specific probes are hybridized with the asymmetric PCR product. In the second protocol (PCR-primer-FP), the fluorescence labeled primer is used in PCR amplification. In both methods, the PCR products are detected using fluorescence polarization. Hybridization (in the PCR-probe-FP method) and conversion (in the PCR-primer-FP method) of 5′-fluorescence labeled oligodeoxynucleotide to the PCR product are monitored by an increase in the anisotropy ratio. The results demonstrate the importance of asymmetric PCR (in the first method) and the selection of dye-modified primer concentration (in the second method) for designing a polarization strategy for the detection of DNA sequence. It has been found that the methods can be used for the identification of infectious agents. This system has also been applied to the determination of Escherichia coli O157:H7 strains. Received: 28 December 1998 / Revised: 22 April 1999 / Accepted: 24 April 1999     

A Single‐Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genomic DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a double‐surface electrochemical technique involving multiple hybridization of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single‐surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen‐printed) electrode. Biotin‐labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidin‐alkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1‐naphthol) which is detected voltammetrically on the same electrode. Signal resulting from this electrochemical enzyme‐linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with osmium tetroxide 2,2′‐bipyridine complex. Detection of (GAA)_n?(TTC)_n triplet repeat expansion in nanogram quantities of PCR‐amplified tDNAs, including amplicons of patients' genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.     

Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells

A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze-thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.     

A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction

A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU mL⁻¹ and 13 CFU mL⁻¹ respectively for these two types of pathogens. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU mL⁻¹ and 25 CFU mL⁻¹, respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrices.     

Enzymatic Synthesis of a DNA Triblock Copolymer that is Composed of Natural and Unnatural Nucleotides

DNA molecules have come under the spotlight as potential templates for the fabrication of nanoscale products, such as molecular‐scale electronic or photonic devices. Herein, we report an enhanced approach for the synthesis of oligoblock copolymer‐type DNA by using the Klenow fragment exonuclease minus of E. coli DNA polymerase I (KF^?) in a multi‐step reaction with natural and unnatural nucleotides. First, we confirmed the applicability of unnatural nucleotides with 7‐deaza‐nucleosides—which was expected because they were non‐metalized nucleotides—on the unique polymerization process known as the “strand‐slippage model”. Because the length of the DNA sequence could be controlled by tuning the reaction time, analogous to a living polymerization reaction on this process, stepwise polymerization provided DNA block copolymers with natural and unnatural bases. AFM images showed that this DNA block copolymer could be metalized sequence‐selectively. This approach could expand the utility of DNA as a template.     

A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.     

Synthesis of water-soluble nucleotide-calixarene conjugates and preliminary investigation of their in vitro DNA replication inhibitory activity

Calix[4]arenes bearing four thymine or adenine 2′-deoxynucleotide moieties have been synthesized and characterized by NMR and ESI-MS analysis. Due to their amphiphilic nature, the conjugates (2a and 2b) obtained tend to self-assemble in aqueous medium by stacking interactions. Their good water solubility makes 2a and 2b promising candidates for bioorganic applications. A preliminary study has provided evidence of their inhibitory activity toward the replication of a Penicillium digitatum DNA fragment via PCR (polymerase chain reaction).