Hexaplex PCR assay and liquid bead array for detection of stacked genetically modified cotton event 281-24-236×3006-210-23

A hexaplex system based on multiplex polymerase chain reaction (PCR) coupled with liquid bead array was developed to assist detection of stacked genetically modified (GM) cotton event 281-24-236 × 3006-210-23 (Widestrike) expressing two kinds of endotoxin from Bacillus thuringiensis (Bt). The efficiency of this multiplex detection system was assessed. Specific primer sets for simultaneous detection of six targets in the stacked GM cotton event were constructed and used for the PCR assay. Each of the six targets was amplified, and the amplicons could be separated as discrete bands by agarose gel electrophoresis. A liquid bead array assay for the stacked GM cotton was performed using the hexaplex PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescent signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescent intensity values. Comparison of the assays showed that results from the liquid bead array using specific probes agreed with those from the PCR, and detection of the different target elements was found to be very specific with no cross-reaction. Therefore, the combination of hexaplex PCR and liquid bead array for detection of stacked GM events can be a useful and efficient system for screening and analyzing multiple transgenes for simultaneous qualitative analysis.     

An allelic ladder based upon reference alleles for mtDNA SNP analysis using the SNaPshot technique

The analysis of mitochondrial DNA (mtDNA) single-nucleotide polymorphisms (SNPs) using the SNaPshot technique (Applied Biosystems) is a fast and sensitive method for the reliable identification of disease-associated mtDNA SNPs, genetic ancestry mtDNA SNPs and forensically important mtDNA SNPs. The detection of many SNPs in one multiplex PCR and one subsequent multiplex minisequencing reaction is challenging for laboratories who want to establish this technique, due to the problem that there is no allelic ladder available for mtDNA SNP analysis via SNaPshot technique. Normally, the laboratory has to invent long-term testing and studies. The interpretation of false and correct alleles is up to some specialists knowing the expected and the estimated size of each allele SNP. We here present a protocol to assemble up to 84 alleles of 42 different mtDNA SNPs in an allelic ladder that is based upon reference alleles. We recommend using allelic ladders/reference alleles for SNP analysis to maintain high-quality analysis standards.     

A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray

Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.     

Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

A simple device for multiplex ELISA made from melt-extruded plastic microcapillary film

We present a simple device for multiplex quantitative enzyme-linked immunosorbant assays (ELISA) made from a novel melt-extruded microcapillary film (MCF) containing a parallel array of 200 μm capillaries along its length. To make ELISA devices different protein antigens or antibodies were immobilised inside individual microcapillaries within long reels of MCF extruded from fluorinated ethylene propylene (FEP). Short pieces of coated film were cut and interfaced with a pipette, allowing sequential uptake of samples and detection solutions into all capillaries from a reagent well. As well as being simple to produce, these FEP MCF devices have excellent light transmittance allowing direct optical interrogation of the capillaries for simple signal quantification. Proof of concept experiments demonstrate both quantitative and multiplex assays in FEP MCF devices using a standard direct ELISA procedure and read using a flatbed scanner. This new multiplex immunoassay platform should find applications ranging from lab detection to point-of-care and field diagnostics.     

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.     

Lateral flow dipstick test for genotyping of 15 beta-globin gene (HBB) mutations with naked-eye detection

For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ∼2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.     

DNA Dangling‐End‐Induced Colloidal Stabilization of Gold Nanoparticles for Colorimetric Single‐Nucleotide Polymorphism Genotyping

A single‐nucleotide polymorphism (SNP) detection method was developed by combining single‐base primer extension and salt‐induced aggregation of gold nanoparticles densely functionalized with double‐stranded DNA (dsDNA‐AuNP). The dsDNA‐AuNPs undergo rapid aggregation in a medium of high ionic strength, whereas particles having a single‐base protrusion at the outermost surface disperse stably, allowing detection of a single‐base difference in length by color changes. When SNP typing primers are used as analytes to hybridize to the single‐stranded DNA on the AuNP surface, the resulting dsDNA‐AuNP works as a visual indicator of single‐base extension. A set of four extension reaction mixtures is prepared using each of ddNTPs and subsequently subjected to the aggregation assay. Three mixtures involving ddNTP that is not complementary to the SNP site in the target produce the aggregates that exhibit a purple color. In contrast, one mixture with the complementary ddNTP generates the single‐base protrusion and appears red. This method could potentially be used in clinical diagnostics for personalized medicine.     

A liquid core waveguide fluorescence detector for multicapillary electrophoresis applied to DNA sequencing in a 91-capillary array

A new laser-induced fluorescence (LIF) detector for multicapillary electrophoresis is presented. The detection principle is based on waveguiding of the emitted fluorescence from the point of illumination to the capillary ends by total internal reflection (TIR) and imaging of the capillary ends. The capillaries themselves thus act as liquid core waveguides (LCWs). At the illumination point, the capillaries are arranged in a planar array, which allows clean and efficient illumination with a line-focused laser beam. The capillary ends are rearranged into a small, densely packed two-dimensional array, which is imaged end-on with high light collection efficiency and excellent image quality. Wavelength dispersion is obtained with a single prism. Intercapillary optical crosstalk is less than 0.5%, and rejection of stray light is very efficient. The detector is applied to four-color DNA sequencing by gel electrophoresis in a 91-capillary array, with simple fluorescein and rhodamine dyes as fluorophores. Since the imaged two-dimensional array is so compact, the detector has a high potential for very large-scale multiplexing.     

Label-free genotyping of single-nucleotide polymorphisms for DNA and RNA targets using a cationic polythiophene derivate

By using the specific primer extension reaction, a new assay for genotyping of single-nucleotide polymorphisms (SNPs) has been demonstrated. The assay relies on the conformational and colorimetric change of water-soluble polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), upon forming interpolyelectrolyte complex with extended double strand DNA and non-extended single strand DNA. All three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. Moreover, combining with the specific digestion of RNA strands in the RNA/DNA hybrids, the proposed assay can also be applied to SNP genotyping for RNA templates. The SNP genotyping assay does not require chemical modification of oligonucleotide probes and nucleic acid targets and any separation step. It would be useful for routinely SNP detection in ordinary laboratories.     

Introduction of an single nucleodite polymorphism-based "Major Y-chromosome haplogroup typing kit" suitable for predicting the geographical origin of male lineages

The European Consortium "High-throughput analysis of single nucleotide polymorphisms for the forensic identification of persons--SNPforID", has performed a selection of candidate Y-chromosome single nucleotide polymorphisms (SNPs) for making inferences on the geographic origin of an unknown sample. From more than 200 SNPs compiled in the phylogenetic tree published by the Y-Chromosome Consortium, and looking at the population studies previously published, a package of 29 SNPs has been selected for the identification of major population haplogroups. A "Major Y-chromosome haplogroup typing kit" has been developed, which allows the multiplex amplification of all 29 SNPs in a single reaction. Allele genotyping was performed with a single base extension reaction (minisequencing) detected by CE. The validation of the multiplex was performed in a total of 1126 unrelated males distributed among 12 worldwide populations. The approach takes advantage of the specific geographic distribution of the Y-chromosome haplogroups and demonstrates the utility of binary polymorphisms to infer the origin of a male lineage.     

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.     

Fluorescence detection system for capillary separations utilizing a liquid core waveguide with an optical fibre-coupled compact spectrometer

A fluorescence detection system for capillary liquid separation methods is described. The system is based on a silica capillary coated with a low refractive index fluoropolymer Teflon AF that serves both as a separation channel and as a liquid core waveguide (LCW). A fibre-coupled laser excites separated analytes in a detection point and arising fluorescence is collected at one end of the LCW capillary into the other optical fibre which brings it to a compact charge-coupled device (CCD) array spectrometer installed in a desktop computer. No additional components such as focusing optics or filters are necessary. This system was used for detecting isoelectrically focused fluorescent low-molecular-mass pI (isoelectric point) markers and fluorescein isothiocyanate (FITC) labelled proteins. The ability of the system to acquire fluorescent spectra is also demonstrated.     

Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.     

Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.     

基于引物延伸反应进行SNP基因分型的电化学方法

引物延伸反应的高特异性使其成为单核苷酸多态性(SNP)基因分型的最常用方法. 本文利用引物延伸反应, 通过二茂铁标记的dUTP将二茂铁引入到延伸的产物中, 用一条捕获探针将延伸产物捕获到电极表面, 用差分脉冲伏安法对电极表面的二茂铁进行检测, 从而实现了SNP基因分型. 考察了延伸反应的退火温度、聚合酶用量以及DNA杂交温度等因素的影响. 应用该方法对β-地中海贫血基因密码子28位单碱基突变进行检测, 获得了满意的基因分型结果. 该方法检测限可达到0.86 fmol/L, 是一种简便、快速且灵敏的SNP分型方法.     

SERS活性液芯光纤的制备及超灵敏检测应用

表面增强拉曼光谱 (SERS)和表面增强共振拉曼光谱 (SERRS)技术的发展使拉曼光谱在各方面的应用突飞猛进 .利用粗糙银电极、蒸镀银岛膜、金和银溶胶的自组装膜等方法制备 SERS活性基底 ,可使拉曼光谱对样品的检测浓度达到 1 0 - 7～ 1 0 - 12 mol/ L,目前可在 1 .0 n L 内检测数十个分子[1～ 3] .1 997年 Nie[4 ] 和 Kneipp等[5] 几乎同时报道拉曼检测达到了单分子水平 .表面修饰的光纤作为传感器 ,在实时、原位或现场检测等应用领域的研究十分活跃 [6～ 9] .液芯光纤作为光纤光谱研究的分支 ,以其在液体样品检测中的独特优势备受关注…