Electrochemical real-time polymerase chain reaction

In this work, we report the first electrochemistry-based real-time polymerase chain reaction technique for sequence-specific nucleic acid detection. This new technique builds upon the advantages of the well-established fluorescence-based counterpart, such as short assay time (simultaneous target DNA amplification and detection). In addition, this electrochemical approach could employ simple and miniaturizable instrumentation compared to the bulky and expensive optics required in the fluorescence-based schemes. We have demonstrated a proof-of-concept experiment showing that the utilization of solid-phase extension of the electrode surface-immobilized capture probe with Fc-dUTP during PCR resulted in the accumulation of the redox marker on the transducer surface. This new technique can be applied to a microfabricated PCR electrochemical device for point-of-care diagnostics as well as on-site environmental monitoring and biowarfare agent detection.     

Conductivity detection of polymerase chain reaction products separated by micro-reversed-phase liquid chromatography

In this paper, we describe the application of micro-reversed-phase high-performance liquid chromatography (mu-RP-HPLC) for the separation and/or purification of polymerase chain reaction (PCR) products with detection accomplished using a miniaturized conductivity detector. The conductivity detector used two Pt wires and a bipolar waveform applied to the electrode pair from which the conductivity of the bulk solution could be measured. In the mobile phase used for the mu-RP-HPLC separation of the PCR product, the mass detection limit for herring sperm DNA using conductivity was found to be 11 ng. Efficient separation of the PCR amplicon from the other reagents present in the PCR cocktail was achieved in less than 4 min with a capacity factor of 2.5 and separation efficiency of 9.1 x 10(3) plates. The separation was carried out using reversed-phase ion-pair chromatography with a triethylammonium acetate ion-pairing agent.     

Aptamer-based polymerase chain reaction for ultrasensitive cell detection

A new system was developed for sensitive and selective detection of tumor cells taking advantage of cell-attached aptamers amplified by PCR and output signals amplified by cationic conjugated polymers.     

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 × 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.     

Electrophoretic human leukocyte antigen HLA-DQA1 DNA typing after polymerase chain reaction amplification.

An electrophoretic method (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is described which permits the identification of human leukocyte antigen HLA-DQA1 types and subtypes without using allele-specific oligonucleotide probes and dot-blot methodology. The procedure can be used in miniaturized gels in combination with automated electrophoretic systems. The PhastSystem is particularly recommended since temperature control is essential. HLA-DQA1*01 and DQA1*0301 can be distinguished in homoduplexes and DQA1*01 subtypes, DQA1*0201 and DQA1*0401 in heteroduplexes (in only 5 h including DNA extraction and PCR amplification). Additional variations to those recognized using commercially available dot-blot methods can be provided since this procedure permits the identification of single base-pair substitutions. In addition, this method is faster and less expensive than commercial methods.     

Real-time polymerase chain reaction detection of fishmeal in feedstuffs

A SYBR Green PCR system was developed for detection of fishmeal in feedstuffs. The real-time PCR method combines the use of fish-specific primers that amplify an 87 base pair (bp) fragment of the mitochondrial 12S ribosomal RNA gene from fish species, and a positive control primer pair that amplifies a 99 bp fragment of the nuclear 18S ribosomal RNA gene in all eukaryotic organisms. The specificity of the primers was tested against 52 animal species and six plant species. Reference feedstuff samples were successfully tested for the presence of fishmeal, demonstrating the applicability of the assay to feedstuffs.     

Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair microcapillary electrochromatography

We describe the development of an integrated microelectrophoretic system consisting of a contact conductivity detector mounted on-chip for monitoring the separation of double-stranded (ds) DNA fragments produced via the polymerase chain reaction (PCR) using microcapillary electrochromatography as the separation mode. The separation was carried out in a polymer-based microfluidic device, hot-embossed into poly(methylmethacrylate) (PMMA), whose walls were functionalized to produce a C(18)-terminated surface to act as the stationary phase (open channel format). The carrier electrolyte contained the ion-pairing agent, triethylammonium acetate (TEAA) to allow the separation to be carried out using reverse-phase ion-pair capillary electrochromatography (RP-IPCEC). The microelectrophoretic separations were investigated utilizing various solvent strengths (acetonitrile/water) with 25 mM TEAA to observe the effects on the separation efficiency as well as the chromatographic development time and detector performance. The field strength significantly affected the quality of the separation, with no separation observed at 333 V/cm for a low mass dsDNA sizing ladder, but baseline separation achieved using a field strength of 67 V/cm. It was observed that the solvent strength affected the retention behavior of the polyanionic molecules as well as the electroosmotic mobility. Higher acetonitrile compositions in the run buffer resulted in reduced plate numbers, which produced lower chromatographic resolution. The use of conductivity detection allowed mass detection sensitivities in the range of 10(-21) mol with a separation efficiency of 10(4) plates and the performance of the detector independent of the acetonitrile content used in the carrier electrolyte.     

Electrochemical genosensor for the detection of interaction between methylene blue and DNA

Described here are the chronocoulometric and voltammetric parameters for methylene blue [3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB] on binding to DNA at carbon paste electrode (CPE) surface. MB, which interacts with the immobilized calf thymus DNA was detected by using single stranded DNA modified CPE (ssDNA modified CPE), bare CPE and double stranded DNA modified CPE (dsDNA modified CPE) in combination with chronocoulometry and differential pulse voltammetry (DPV) techniques. The effect of ionic strength to the behavior of MB with dsDNA and ssDNA was also studied by means of voltammetry. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.     

Ferrite supports for isolation of DNA from complex samples and polymerase chain reaction amplification

The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR.     

Ligase chain reaction amplification for sensitive electrochemiluminescent detection of single nucleotide polymorphisms

Single nucleotide polymorphisms are the most common type of genetic variations among human beings and can serve as biomarkers for various types of diseases. In this work, based on ligase chain reaction amplification for the production of massive hemin/G-quadruplex DNAzymes to quench the electrochemiluminescent (ECL) emission of quantum dots (QDs), a universal and sensitive single nucleotide polymorphism detection method is described. During the ligase chain reaction process, the mutant K-ras target gene is recycled and exponentially duplicated, leading to the attachment of numerous G-rich sequences on the QD-embedded sensing surface. Upon the addition of the assistant sequences and hemin, numerous hemin/G-quadruplex DNAzymes are formed, which consume the dissolved oxygen in the detection buffer and result in significant quenching of QD ECL emission for sensitive single nucleotide polymorphism determination. The developed method shows a linear range of 50 fM to 50 pM and an estimated detection limit of 45 fM for the mutant K-ras gene. The proposed strategy also exhibits high selectivity towards the mutant K-ras gene against the co-existence of 10³-fold excess of the wild-type K-ras gene, which makes our method a useful addition to the alternatives for single nucleotide polymorphism monitoring.     

Species-specific polymerase chain reaction amplification of camel (Camelus) DNA extracts

A sensitive polymerase chain reaction (PCR) method based on amplification of a specific DNA fragment was established for the identification of camel (Camelus) materials. The species-specific primer pair L183/H372 was designed based on the nucleotide sequence of the mitochondrial cytochrome b gene, and its specificity was confirmed by amplification of 3 camel (domestic double-humped camel, wild double-humped camel, wild one-humped camel) samples and 11 non-Camelus animal (sheep, goat, pig, chicken, cattle, fish, dog, horse, donkey, deer, and rabbit) materials. An expected 208 base pair fragment was amplified from camel materials; no cross-reactive or additional fragments were generated from other animal materials. Taq I restriction endonuclease digestion of the unpurified PCR product can be used routinely to confirm the camel origin of the amplified sequence.     

Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction

In this report, Au-coated nanostructured biochip with functionalized thiolated primers on its surface is developed for label-free and real-time optical detection of polymerase chain reaction (PCR). A PCR chamber of 150 μm in thickness containing Au-coated nanostructured substrate in the bottom layer was bordered with SU-8 100 walls. After immobilization of 5′-thiolated primers on the surface, simultaneous DNA amplification and detection were performed without any labeled molecules via the relative reflected intensity (RRI) of Au-coated nanostructured substrate. When human genomic DNA at several concentrations of 0.2, 0.5 and 1 ng μL⁻¹ was included in the initial DNA samples, the increases in the RRI peak values were clearly observed with the increasing PCR cycle numbers. We found that the starting point of the optical signal, which was divergent from the background in our PCR biochip, was around 3-4 cycles, much lower than that of the fluorescent real-time PCR analysis (around 23-25 cycles). Our proposed PCR device using Au-coated nanostructured substrate holds noteworthy promise for rapid, label-free and real-time DNA detection for point-of-care testing (POCT) applications.     

Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.     

Multiple use of slab gels in sequencing apparatus for separation of polymerase chain reaction products

Attempting to assess whether a decrease of the electrophoresis temperature could prevent or reduce the extent of gel well deformations, and whether the utilization of native polyacrylamide gels (without urea) could speed up the separation of polymerase chain reaction (PCR)-amplified products with an automated 377 DNA sequencer, denatured PCR products were subjected to electrophoresis in 6% native gels under 45 degrees C. Results show that a decrease of the electrophoresis temperature from 51 degrees C (recommended by the User's Manual) to 45 degrees C substantially facilitates the preservation of gel wells, and that all PCR products tested migrate significantly faster in native than in denatured (with urea) gels of the same concentration. The combination of a 6% native gel and a lower (45 degrees C) electrophoresis temperature permits multiple uses of a given gel with consistent results, consequently reducing the electrophoresis time and reagent costs.     

An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40 × 40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.     

Evaluation of the LTQ-Orbitrap mass spectrometer for the analysis of polymerase chain reaction products

We have investigated the potential and robustness of the off‐line coupling of polymerase chain reaction (PCR) with electrospray ionization mass spectrometry (ESI‐MS), for further applications in the screening of single‐nucleotide polymorphisms (SNPs). This was based on recently reported data demonstrating that anion‐exchange solid‐phase extraction was the most efficient technique for efficiently desalting PCR products, with a recovery of ～70%. Results showed that this purification approach efficiently removes almost all the chemicals commonly added to PCR buffers. ESI‐MS analysis of a model 114‐bp PCR product performed on the LTQ‐Orbitrap instrument demonstrated that detection limits in the nM range along with an average mass measurement uncertainty of 9.15 ± 7.11 ppm can be routinely obtained using an external calibration. The PCR/ESI‐MS platform was able to detect just a few copies of a targeted oligonucleotide. However, it was shown that if two PCR products are present in a mixture in a ratio higher than 10 to 1, the lower abundance one might not be reproducibly detected. Applications to SNPs demonstrated that an LTQ‐Orbitrap with a resolution of 30 000 (at m/z 400) easily identified a single (A ? G) switch, i.e. a 16 Da difference, in binary mixtures of ～ 35 kDa PCR products. Complementary experiments also showed that the combination of endonucleases and ESI‐MS could be used to confirm base composition and sequence, and thus to screen for unknown polymorphisms in specific sequences. For example, a single (T ? A) switch (9 Da mass difference) was successfully identified in a 114‐bp PCR product. Copyright © 2010 John Wiley & Sons, Ltd.     

An improved electrochemiluminescence polymerase chain reaction method for the detection of Fusarium wilts

An improved electrochemiluminescence polymerase chain reaction （ECL-PCR） method was developed and applied to detect Fusarium wilt. Briefly, the internal transcribed spacer （ITS） sequence of Fusarium oxysporumf, sp Cubense （FOC） was amplified by PCR. Two universal fragments, which were complimentary to Ru（bpy）3^2＋ （TBR） labeled probe and Biotin labeled probe, respectively, were connected to the tail of primers so that all the PCR products got universal sequences. Then biotin labeled probes and TBR labeled probes were hybridized with the PCR products at the same time. Through the specific interaction between biotin and streptavidin, the PCR products were captured by streptavidin coated magnetic bead and then detected by ECL assay. The experiment results showed that the healthy banana samples and infected ones can be discriminated by this ECL-PCR method. This improved ECL-PCR approach is useful in Fusarium wilt detection due to its high sensitivity, simplicity and stability.     

The use of arbitrarily primed polymerase chain reaction in cancer research

There is a great need for techniques that detect the genome alterations present in cancer cells. Here, we present a review of the arbitrarily primed polymerase chain reaction (AP-PCR), a genomic mutation detection method with some unique advantages: (i) It can detect most types of mutations that usually occur in tumors (except point mutations). (ii) It is especially useful to detect moderate gains in DNA, which most methods currently in use cannot detect. (iii) It allows detection and cloning of alterations in a single experiment. (iv) It is inexpensive and does not require special equipment. We discuss some characteristics of this method and review some of its achievements in cancer research.