实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.     

Numerical simulation of DNA sample preconcentration in microdevice electrophoresis

A numerical model is presented for the accurate and efficient prediction of preconcentration and transport of DNA during sample introduction and injection in microcapillary electrophoresis. The model incorporates conservation laws for the different buffer ions, salt ions, and DNA sample, coupled through a Gaussian electric field to account for the field modifications that cause electromigration. The accuracy and efficiency required to capture the physics associated with such a complex transient problem are realized by the use of the finite element-flux corrected transport (FE-FCT) algorithm in two dimensions. The model has been employed for the prediction of DNA sample preconcentration and transport during electrophoresis in a double-T injector microdevice. To test its validity, the numerical results have been compared with the corresponding experimental data under similar conditions, and excellent agreement has been found. Finally, detailed results from a simulation of DNA sample preconcentration in electrophoretic microdevices are presented using as parameters the electric field strength and the other species concentrations. The effect of the Tris concentration on sample stacking is also investigated. These results demonstrate the great potential offered by the model for future optimization of such microchip devices with respect to significantly enhanced speed and resolution of sample separation.     

Multiplexed microRNA detection by capillary electrophoresis with laser-induced fluorescence

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.     

Flow-through sampling for electrophoresis-based microfluidic chips using hydrodynamic pumping

This work presents a novel electrophoretic microchip design which is capable of directly coupling with flow-through analyzers for uninterrupted sampling. In this device, a 3 mm wide sampling channel (SC) was etched on quartz substrate to create the sample inlet and outlet and the 75 microm wide electrophoretic channels were also fabricated on the same substrate. Pressure was used to drive the sample flow through the external tube into the SC and the flow was then split into outlet and electrophoretic channels. A gating voltage was applied to the electrophoretic channel to control the sample loading for subsequent separations and inhibit the sample leakage. The minimum gating voltage required to inhibit the sample leakage depended on the solution buffer and increased with the hydrodynamic flow-rate. A fluorescent dye mixture containing Rhodamine B and Cy3 was introduced into the sample stream at either a continuous or discrete mode via an on-line injection valve and then separated and detected on the microchip using laser-induced fluorescence. For both modes, the relative standard deviation of migration time and peak intensity for consecutive injections was determined to be below 0.6 and 8%, respectively. Because the SC was kept floating, the external sampling equipment requires no electric connection. Therefore, such an electrophoresis-based microchip can be directly coupled with any pressure-driven flow analyzers without hardware modifications. To our best knowledge, this is something currently impossible for reported electrophoretic microchip designs.     

On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology

Integrating cell lysis and DNA purification process into a micrototal analytical system (microTAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20 min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system.     

High throughput genetic screening for the detection of hereditary non-polyposis colon cancer (HNPCC) using capillary electrophoresis

Approximately 5-10% of all colorectal carcinomas arise from cancer predisposition syndromes caused by heterozygote germline mutations in post-replicative DNA mismatch repair (MMR) genes. In contrast to gastrointestinal polyposis syndromes, carcinomas in these patients do not occur on the background of increased numbers of polyps and hence are refered to as hereditary non-polyposis colorectal cancers (HNPCC). Six different MMR genes, MSH2, MSH3, MSH6, MLH1, MLH3 and PMS2, have been identified in the human genome. In the majority of HNPCC patients, heterozygote germline mutations are present in the MSH2 or MLH1 gene. Detection of mutations by conventional sequencing technology is expensive and labor intensive due to the complex intron and/or exon structures. In this study, we therefore have explored whether capillary electrophoresis-based single strand conformation polymorphism (SSCP-CE) provides a reliable means for mutation screening. We have tested different MLH1 mutations in exons 9 and 16 and find that SSCP-CE produces reliable electrophoretic patterns that allow recognition of wild-type alleles, microdeletions and point mutations. In summary, SSCP-CE provides a rapid, automated, and cost-effective technology for MSH2 and MLH1 mutation screening and will facilitate genetic diagnostics for HNPCC patients.     

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines

The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists in protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yet the 2-D PAGE-mass spectrometry (MS) approach has its drawbacks with regard to automation, sensitivity, and throughput. Consequently, considerable effort has been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings of 2-D PAGE. In addition, traditional Chinese medicines (TCMs), due to their long period of clinical testing and reliable therapeutic efficacy, are attracting increased global attention. However, hundreds or even thousands of components are usually present in TCMs, which results in great difficulties of separation. As a mainstream separation tool, multidimensional liquid separation systems have shown powerful separation ability, high peak capacity, and excellent detectability in the analysis of complex samples including biological samples and TCMs, etc. Therefore, this review emphasizes the most recent advances in multidimensional liquid chromatography and capillary electrophoresis-based separation techniques, and the corresponding applications in proteomics and TCMs. In view of the significant contributions from Chinese scientists, this review focuses mainly on the work of Chinese scientists in the above fields.     

Further improvement of hydrostatic pressure sample injection for microchip electrophoresis

Hydrostatic pressure sample injection method is able to minimize the number of electrodes needed for a microchip electrophoresis process; however, it neither can be applied for electrophoretic DNA sizing, nor can be implemented on the widely used single-cross microchip. This paper presents an injector design that makes the hydrostatic pressure sample injection method suitable for DNA sizing. By introducing an assistant channel into the normal double-cross injector, a rugged DNA sample plug suitable for sizing can be successfully formed within the cross area during the sample loading. This paper also demonstrates that the hydrostatic pressure sample injection can be performed in the single-cross microchip by controlling the radial position of the detection point in the separation channel. Rhodamine 123 and its derivative as model sample were successfully separated.     

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension)

Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch? method. Notably, pathogen detection via IFAST required only 8.5 μL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.     

On-line sample preconcentration in capillary electrophoresis, focused on the determination of proteins and peptides

This overview highlights the possibilities of on- or in-line preconcentration procedures in combination with a CZE separation, focused on the determination of peptides and proteins. The discussed methods, including sample stacking, field-amplified injection, isotachophoresis, solid phase extraction, membrane preconcentration, electroextraction, supported liquid membranes, hollow fibers, immunoaffinity, and molecularly imprinted polymers technology preconcentration are categorized in electrophoresis-based and chromatography-based preconcentration. The chromatography-based preconcentration is subdivided in low-specificity and high-specificity methods. A number of preconcentration methods are available, however, this paper demonstrates that various compounds in different media (aqueous solutions, urine, and plasma) require different preconcentration systems. The preconcentration techniques of first choice in general seem to be solid-phase extraction and membrane preconcentration, because of their high concentration ability, multiapplicability, relative simplicity and clean-up capability. For the future, hollow fibers seem to hold a great potential as preconcentration technique, yielding high concentration factors, using simple designs. New techniques, such as hollow fibers, molecularly imprinted polymers technology and supported liquid membranes may have the potential to supersede the conventional preconcentration techniques in some cases. The larger the arsenal of preconcentration techniques becomes, the more efficiently peptides and proteins may be analyzed in the future. These techniques, in some cases, require pre-cleanup procedures, to ensure the purity of the samples to concentrate.     

Probing affinity via capillary electrophoresis: advances in 2003–2004

This review addresses recent advances in capillary electrophoresis of biological-based molecular interaction from a broader perspective, based on applications reported during the period 2003–2004. These capillary electrophoresis-based studies of molecular interactions include affinity capillary electrophoresis, electrokinetic chromatography, and free zone electrophoresis. The review is written as a general synopsis of applications and does not cover the theory or protocol involved in the implementation of the analyses.The caption to Fig. 3 was incorrect. This revised version replaces the article published online on 10 February 2005.     

Capillary electrochromatography using polyelectrolyte multilayer coatings

This review covers recent progress in polyelectrolyte multilayer (PEM) coatings applied to analytical separations using open-tubular capillary electrochromatography (OT-CEC). The simple preparation procedure involved in the PEM approach has provided some attractive features over other modes of capillary electrophoresis-based separations including packed column capillary electrochromatography (PC-CEC) and micellar electrokinetic chromatography (MEKC). PEM coatings have been used to alleviate the adsorption of basic analytes, to improve separations, and to improve the stability of the electroosmotic flow. Fundamental aspects of PEM coatings on surfaces and analytical separation platforms are briefly outlined in this review. In addition, applications of PEM coatings to fused-silica capillaries or microchip separation devices for the separation of small achiral or chiral analytes, as well as large biomolecules, are discussed.     

A method for DNA detection in a microchannel: fluid dynamics phenomena and optimization of microchannel structure

A simple DNA diagnosis method using microfluidics has been developed which requires simple and straightforward procedures such as injection of sample and probe DNA solutions. This method takes advantage of the highly accurate control of fluids in microchannels, and is superior to DNA microarray diagnosis methods due to its simplicity, highly quantitative determination, and high-sensitivity. The method is capable of detecting DNA hybridization for molecules as small as a 20 mer. This suggests the difference in microfluidic behavior between single strand DNA (ssDNA) and double stranded DNA (dsDNA). In this work, influence of both the inertial force exerted on DNA molecules and the diffusion of DNA molecules was investigated. Based on the determination of these parameters for both ssDNA and dsDNA by experiments, a numerical model describing the phenomena in the microchannel was designed. Computational simulation results using this model were in good agreement with previously reported experimental results. The simulation results showed that appropriate selection of the analysis point and the design of microchannel structure are important to bring out the diffusion and inertial force effects suitably and increase the sensitivity of the detection of DNA hybridization, that is, the analytical performance of the microfluidic DNA chip.     

Detection of genetically modified crops and their derivatives: critical steps in sample preparation and extraction

The detection of genetically modified crops in foodstuff relies on detection of transgenic DNA or protein material in the sample matrix. Purified DNA or proteins are used as analytical material for polymerase chain reaction technologies and immunodiagnostics. Successful sample preparation is critical to the validity of subsequent analysis. For routine analysis, a good sample preparation technique should be simple, safe, and inexpensive while reproducibly generating DNA/protein of sufficient quality and yield. The suitability of isolated DNA or protein as an analyte for a detection or characterization technique depends on amount or concentration, purity, and integrity, each of which may be influenced by sample matrix and the extraction technique, and, in turn, may impact the validity of analytical techniques. The key sample preparation steps of homogenization, pretreatment, extraction, and purification are discussed as well as typical analytical methods. Consideration is given to application of these steps for particular sample matrixes to maximize yield, reduce inhibition effects, and minimize contamination. The choice of the most appropriate and valid methods for sample preparation from particular foods is discussed with respect to DNA analysis. Attention is also given to ease of use, cost, and generic applicability of the procedures.     

Quantitative polymerase chain reaction using capillary electrophoresis with laser-induced fluorescence detection: Analysis of duck hepatitis B

We report an accurate and reproducible DNA quantitation method using the polymerase chain reaction (PCR). The amount of PCR product is monitored after each PCR cycle by capillary electrophoresis. To ensure accurate quantitation, a non-amplified internal standard is added to each PCR-amplified electrophoresis sample to correct for variations in injection volume. Quantitation of the sample is based on the number of cycles necessary to generate a predetermined amount of PCR product. Duck hepatitis B virus genome was used as a model in this study. The genome was quantified with a linear relationship between cycle number and logarithm of sample DNA for amounts of sample DNA between 30 and 3.1 x 10(8) copies ( r(2)>0.999). The relative standard deviation for the corrected capillary electrophoresis signal was 2.7%, while the relative standard deviation for the overall assay was 3.0%. Results from a single-blind study generated a relative error of 1.3%.     

电化学发光PCR定量检测H-ras癌基因点突变

提出了一种电化学发光PCR(ECL-PCR)分析方法,该法可用于定量检测基因点突变.其检测H-ras癌基因PCR扩增产物的灵敏度可达100fmol;线性范围为0.1-500pmol.用ECL-PCR分析法对膀胱癌组织中H-ras癌基因进行突变检测,只需要10μL样品,20min的孵育时间和30s的采集时间,得出20例膀胱癌样品中有7例存在点突变,通过标准曲线方程定量计算出突变样品的量.ECL-PCR分析方法在灵敏度、线性范围、分析时间等方面都优于传统的检测方法,是一种安全、快速、灵敏、定量检测基因点突变的分析方法.     

Single Molecule Assay of Escherichia coli β-Galactosidase Using Two Competing Substrates Simultaneously,DDAO-β-D-Galactoside and Resorufin-β-D-Galactoside

Single enzyme molecule assays were performed on E. coli β-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-β-D-galactoside and DDAO-β-D-galactoside, simultaneously. The variation between individual enzyme molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given enzyme molecule. This suggests that the enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.     

Proteome analysis using isoelectric focusing in immobilized pH gradient gels followed by mass spectrometry

Over the past several years, a large effort has been focused on improvements of two-dimensional (2-D) gel electrophoresis-based proteomics technology, and on development of novel approaches for proteome analysis. Here, we describe the application of an alternative strategy for the analysis of complex proteomes. The strategy combines isoelectric focusing in immobilized pH gradient strips (in-gel IEF), mass spectrometry (MS), and bioinformatics. A protein mixture is separated by in-gel IEF, and the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the tryptic peptides are subjected to liquid chromatography-nanoelectrospray-quadrupole ion-trap tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS data are used to identify the proteins through searches of a protein sequence database. Using this in-gel IEF-LC-MS/MS strategy, we have identified 127 proteins from a human pituitary. This study demonstrates the potential of the in-gel IEF-LC-MS/MS approach for analyses of complex mammalian proteomes.     

Macro-to-micro interfaces for microfluidic devices

Since the concept of miniaturized total analysis systems (microTAS) was invented, a great number of microfluidic devices have been demonstrated for a variety of applications. However, an important hurdle that still needs to be cleared is the connection of a microfluidic device with the rest of the world, which is often referred to as the macro-to-micro interface, interconnect, or world-to-chip interface. In this review, we will examine the methods used by pioneers in the field and other investigators, review the approaches for capillary electrophoresis-based devices and those using pneumatic pumping, and present additional discussion on interface standardization and choosing and designing interconnects for your applications.