基于自然对流PCR与毛细管电泳技术的牙周病原菌检测

细菌种类的快速识别在牙病防治中具有重要的应用价值。以牙周病原菌为研究对象,建立了DNA快速扩增的自然对流聚合酶链式反应(PCR)方法及毛细管电泳荧光光学检测系统。研究表明,当毛细管有效长度为8cm、电场强度为100V/cm时,50bp DNA ladder在筛分介质为0.5%HEC(羟乙基纤维素)(1 300K)中的分离效果最佳。毛细管电泳结果表明,采用自然对流法可以在25min内在圆柱腔体中实现牙周病原菌的快速PCR。     

STR分型检测仪器性能评价方法研究

目的:针对自主研制的GA118-16A遗传分析仪,设计一系列的检测方法以实现对仪器性能的评价。方法:从仪器的迁移速度、灵敏度、重复性、分辨力等方面进行检测方法设计,并进行试验检验。结果:以Gene Scan-500 LIZ为标准物质,根据迁移速度与电场强度关系,在电泳电压大于13KV时可在规定时间内完成500bp片段长度电泳,并进行有效分离,RSD≤2%;仪器的最低上样检测灵敏度可达3.8×10-2ng/μL;能准确分辨1bp差异的DNA片段;重复性RSD≤0.8%。结论:通过设计的实验方法可以对该分析仪的迁移速度、灵敏度等各项性能指标进行较好的评价。     

荧光标记DNA分子量内标的设计与制备

目的:设计并制备65bp-500bp范围的荧光标记DNA分子量内标。方法:p MD18-T Vector连接任意一段割胶回收的DNA片段,克隆后提取重组质粒经双酶切后作为模板,在其上游设计一条固定引物并用荧光标记,在其下游设计一系列引物,经PCR扩增后在ABI 3100遗传分析仪上检测。结果:扩增产物DNA片段大小分别为65bp、105bp、149bp、200bp、241bp、269bp、311bp、345bp、400bp、450bp、500bp,与预期片段大小一致。结论:11个片段经混合调平后,峰型良好,出峰位置均匀,可用于毛细管电泳中DNA片段大小的确定。     

多通道DNA测序仪的新型设计

目前大多数商品化的DNA测序仪的信号检测系统是基于CCD或PMT.基于CCD的系统需要对CCD进行制冷,其造价相对较高且灵敏度相对较低;基于PMT的系统使用机械扫描来实现多通道检测,其工作稳定性相对较低且工作的机械噪音大.本文提出了新型的DNA测序仪,它采用PMT共焦荧光系统,通过f-θ透镜和光学扫描来实现多通道并行检测.在此新系统中采用标准双链DNA样品pBR322/Hae Ⅲ(使用TO染料)进行了毛细管电泳实验,测得系统的检测限可达1.1841 × 10-11 mol/L.新型DNA测序仪的工作机械噪声相比基于机械扫描的系统要低得多,且工作稳定性和灵敏度也很高.此系统可应用于基于激光诱导荧光的毛细管阵列和多通道微芯片的电泳检测.     

低电压驱动毛细管电泳芯片电势分布的数值计算

低电压驱动阵列电极式毛细管电泳芯片是针对毛细管电泳芯片分离电压高、系统体积庞大的缺点,在原有芯片的结构上进行改进的电泳芯片.通过建立数学模型,计算了设置阵列电极后毛细管电泳微沟道内的电势分布,并与常规进样条件下微沟道内的电势分布进行比较,结果表明该方法可以使分离电压大幅度下降.     

聚砜中空纤维膜接口的改进及其在二维毛细管电泳蛋白质分离平台构建中的应用

介绍了一种在自制的聚砜中空纤维膜基础上改进接口的连接方法,并将其用于构建二维毛细管电泳蛋白质分离技术平台.经改进的中空纤维膜接口具有死体积小、传质效率高、分析速度快等优点,是目前多维毛细管电泳较为理想的接口.本文以细胞色素C为标样对二维毛细管等电聚焦和毛细管区带电泳的分离性能进行了考察,同时对肺癌细胞的分泌蛋白进行了分离分析.实验结果表明与一维系统分离结果比较二维毛细管电泳在分辨率和峰容量方面都有较大的提高.     

智能化多功能毛细管电色谱仪的研制

研制出一种智能化毛细管电色谱仪,介绍了该仪器的工作原理,构成和特点。设计了一种包括具有极小死体积的进样器,恒压分离系统和适于梯度洗脱的分流器的流路结构,可有效地解决CEC现存的主要问题,仪器的自动控制系统基于Intel80C196单片机,包括A／D,D／A转换和串口通信等模块,控制系统通过串行口与PC机通信,读取用户设置的参数和程序,完成对高压电源和泵的控制,在同一台仪器上可实现电压驱动电色谱,压力驱动电色谱,微柱液相色谱和毛细管电泳等多种分离模式。测试了该仪器的分离重现性和自动控制的可靠性,获得满意结果。该仪器采用梯度加压毛细管电色谱已成功地用于10条DNA片段分离。     

毛细管电泳的离子富集性能研究

毛细管电泳是一种高效的离子富集分离技术。利用毛细管区带电泳法,背景电解质选用浓度为10mmol/L的咪唑,选择性改性剂使用浓度为10mmol/L的α-羟基乙酸,pH值控制在4.0、选用20kV的分离电压、分离温度为室温25℃。在优化的电泳条件下,几种金属离子在15min内可得到有效分离,且具有较低的检出限和良好的线性关系。选用适合的背景电解质和选择性改性剂,在优化的试验条件下可以有效解决目前生物分析、食品卫生等领域痕量元素分析的需求。     

21世纪毛细管电泳在刑侦分析中的应用

介绍了毛细管电泳的分离机理和方法分类;评述了2000～2009年毛细管电泳法在刑侦分析中的应用,重点是在DNA分析方面的应用;对未来发展做了讨论,引用文献49篇。     

微通道电泳芯片系统中信号识别算法的设计与实现

信号处理与分析软件是微全分析系统的重要组成部分，也是生物信息学研究的一个重要领域。本文介绍了适用于微通道电泳芯片系统的DNA测序软件的总体流程，重点描述了其中的信号处理与识别算法。算法以激光诱导荧光检测系统所采集到的原始数据为源信号，以小波平滑和小波去噪为理论基础，将滤波算法和峰值识别算法综合在一起进行设计，从而使其适用于检测速度更快、样品量更少的微通道电泳芯片系统。将本算法应用于DNA片段的分离实验中，可以有效地达到滤波以及信号识别的目的。     

Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer

The measurement by atomic force microscope of the contour length of DNA fragments adsorbed on mica has been made as accurate as possible by revisiting the different steps of image acquisition and processing. In air, the DNA helical rise was estimated at 2.97 +/- 0.15 A per base pair (bp) (mean +/- standard deviation) by imaging a 648-bp DNA fragment and 2.95 +/- 0.14 A per bp for a 115-bp fragment. This confirms earlier observations suggesting that drying DNA fragments on mica in the presence of nickel induces limited conformational changes. At this point the exact nature of these conformational changes remains unknown. Simple hypotheses are the transconformation of stretches of the DNA molecules to the A-form of the double helix or alteration of the helix structure at the points of contact between DNA and mica. By contrast, in aqueous buffer, the measured helical rise was 3.14 +/- 0.15 A per bp for the 648-bp fragment and 3.17 +/- 0.13 A per bp for the 1115-bp fragment. Thus, measured helical rises do not depend on the fragment length and are significantly shorter than the 3.38 A per bp measured by crystallography, but close to the 3.18 A per bp found in NMR studies. These findings are discussed with respect to discrepancies in earlier results published in the literature.     

Design and operation of a portable scanner for high performance microchip capillary array electrophoresis

We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ~20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex(?) 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.     

毛细管电泳法检测化妆品中对羟基苯甲酸酯类防腐剂

采用毛细管区带电泳法同时测定化妆品中4种对羟基苯甲酸酯类防腐剂。色谱条件是:75 μm× 57 cm(有效长度50 cm)毛细管,电解质为25 mmol/L硼砂(pH 10.0)缓冲溶液,分离电压20kV,温度25℃,0.5 psi进样5s,检测波长200 nm。对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯在10 min内基线分离,10～500 μg/mL范围内线性关系良好(r>0.9949),检出限为0.60～1.20 μg/mL,样品加标回收率为75.5%～112.0%。     

基于ARM9的共聚焦式激光诱导荧光检测系统

共聚焦式激光诱导荧光检测具有较高的灵敏度,是毛细管电泳芯片的主要检测方法之一。大多数共聚焦式检测系统采用计算机控制和显示,系统体积庞大,无法实现小型化和集成化。文中基于ARM9微处理器及嵌入式Linux操作系统,设计了脱离计算机控制的共聚焦式毛细管电泳芯片检测系统,实现了芯片的图像观察、自动聚焦及荧光信号采集等功能。系统对不同浓度的罗丹明B样品进行了电泳分离实验,检测限为1.0×10-7mol/L.     

Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.     

CAPILLARY ELECTROPHORETIC SEPARATION OF DNA FRAGMENTS UNDER STEPWISE CHANGES OF POLYMER SOLUTIONS

ABSTRACT

We demonstrated techniques of stepwise changes of poly-ethyleneoxide (PEO) solutions for DNA separations in the presence of the electroosmotic flow (EOF). The stepwise changes were performed by injecting different PEO solutions contained in a number of syringes to a polyethylene tube, from which the polymer solutions entered a capillary filled with 1X TBE buffer by the EOF. The separation of DNA markers V and VI was not complete under isocratic conditions, either using 1.0–2.5% PEO solutions containing 0.5 μg/mL ethidium bromide (EtB) or using 2.0% PEO solutions containing 0.1–2.0 μg/mL EtB. Resolution, sensitivity and time of the DNA separation were varied under conditions of either stepwise changes of PEO concentrations from 1.5 to 2.5% in 0.5 μg/mL EtB solutions or stepwise changes of EtB concentrations from 0.1 to 2.0 μg/mL in 2.0% PEO solutions.

The optimal separation was obtained under simultaneous stepwise changes of PEO and EtB: 1.5% PEO containing 0.1μg/mL EtB for 45 s, 1.5% PEO containing 0.5μg/mL EtB for 120 s, 2.0% PEO containing 0.5μg/mL EtB for 45 s, 2.0% PEO containing 1.0μg/mL EtB for 30 s, and 2.5% PEO containing 2.0μg/mL EtB for the rest. The separation was complete in 17 min and the RSD values of migration times were less than 2.0%.     

Patterning DNA on microm scale on mica

Double-stranded DNA molecules were patterned by selective adsorption to aminosilane patterns on mica surfaces. Line patterns with 10 microm spacing were made by photolithography and transferred to a polymer stamp. The stamp was then used for applying aminosilane molecules by microcontact-printing technique on mica substrates. We applied DNA in Tris-EDTA (TE) buffer solution on the patterned substrate, and incubated it for 5 min at room temperature. The sample was then rinsed with pure water, and dried with nitrogen gas. Tapping mode force microscopy showed that DNA was adsorbed selectively on the aminosilanized parts of the mica substrate. We also tried to bridge two aluminum electrodes with DNA using AC electrophoresis.