基于荧光显微镜的毛细管电泳系统的构建

毛细管电泳仪具有灵敏度高、分析速度快等优势,为降低其生产成本,基于电泳原理,以荧光显微镜为基础,设计了一套毛细管电泳系统。以20 bp(base pairs,碱基对)DNA ladder和100 bp DNA ladder为样本,全面分析了系统的稳定性、灵敏度和分离效果。结果表明:该系统在9 min内可以实现1500 bp以内DNA片段的高效分离,系统检测极限为0.1 ng/μL;在优化的电泳条件下,对限制性内切酶φX174-HincⅡ作用过的λ-DNA片段5 min内实现了291 bp与297 bp DNA片段的区分。     

In-Fusion克隆技术构建HBV X基因真核表达质粒

目的 :以血清中乙型肝炎病毒(HBV)DNA为模板,克隆构建HBV X蛋白质的真核表达载体pcDNA-HBx。方法 :设计扩增HBV X基因的In-Fusion PCR引物,采用高保真DNA聚合酶分两段扩增HBV X基因序列;采用In-Fusion克隆技术,将两段X基因扩增片段一步克隆入经Hind III和Xba I双酶切的pcDNA3.0真核表达载体,以构建重组真核表达载体pcDNA-HBX;采用Hind III和Xba I双酶切和DNA序列测序筛选鉴定重组载体;pcDNAHBx转染HepG2细胞,Western blot检测pcDNA-HBx转染细胞的HBx蛋白质表达。结果 :InFusion PCR引物分两段扩增获得全长HBx基因序列;In-Fusion获得克隆的pcDNA-HBx经双酶切筛选得到阳性重组质粒,测序分析证实插入序列正确;转染pcDNA-HBX的HepG2细胞,Western blot证实表达HBx蛋白质。结论 :以血清HBV DNA为模板克隆HBV X基因,构建了表达HBV HBx蛋白质的真核表达载体,为HBx蛋白质的生物学功能研究提供支持。     

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。     

电极优化的PCR芯片及其扩增实验

对传统的蛇形加热电极结构进行了优化,提升PCR芯片反应的温度分布均匀性。设计了一种电极片可重复使用且和反应腔室片可分离的PCR芯片。利用红外热像测温仪对优化前后的电极片进行了温度场分布测量,结果表明优化后的加热电极在反应腔室范围内的温度均匀性小于1℃。与大型PCR仪的扩增实验结果对比表明,该PCR芯片实现了239 bp的DNA片段的高效扩增。     

In-Fusion克隆技术构建HBVX基因真核表达质粒

目的以血清中乙型肝炎病毒（HBV）DNA 为模板,克隆构建HBV X 蛋白质的真核表达载体pcDNA-HBx.方法：设计扩增HBV X 基因的In-Fusion PCR 引物,采用高保真DNA 聚合酶分两段扩增HBV X 基因序列;采用In-Fusion 克隆技术,将两段X 基因扩增片段-步克隆入经Hind Ⅲ 和Xba I 双酶切的pcDNA3.0 真核表达载体,以构建重组真核表达载体pcDNA-HBX ;采用Hind Ⅲ 和Xba I 双酶切和DNA 序列测序筛选鉴定重组载体;pcDNAHBx转染HepG2 细胞,Western blot 检测pcDNA-HBx 转染细胞的HBx 蛋白质表达.结果：In-Fusion PCR 引物分两段扩增获得全长HBx 基因序列;In-Fusion 获得克隆的pcDNA-HBx 经双酶切筛选得到阳性重组质粒,测序分析证实插入序列正确;转染pcDNA-HBX 的HepG2 细胞,Western blot 证实表达HBx 蛋白质.结论：以血清HBV DNA 为模板克隆HBV X 基因,构建了表达HBV HBx 蛋白质的真核表达载体,为HBx 蛋白质的生物学功能研究提供支持.     

应用新型荧光定量PCR技术检测FGFR2基因SNP

目的:探讨一种目前较为新颖的通过Rotor Gene-3000荧光定量PCR仪进行Tm-shifting荧光定量PCR扩增测定单核苷酸多态性(SNP)的可行性。方法:以913份乳腺肿物患者成纤维细胞生长因子受体2(FGFR2)基因rs2981582位点(T→C)作为研究对象,用荧光染料SYBR GreenI标记DNA,并在设计引物时在所研究基因的特异性引物5’末端连接上不同长度的尾,使不同基因型标本的PCR融解曲线的高峰出现在不同位置,最终可通过对PCR融解曲线的分析进行SNP分型测定。结果:纯合子C/C基因型融解曲线峰出现在≤84.60C处,特殊情况下稍向右偏移,但不超过85.10C,峰型较尖。纯合子T/T基因型融解曲线峰出现在≥87.50C处,特殊情况下稍向左偏移,但不小于870C,峰型较尖。杂合子T/C基因型融解曲线峰出现在前两者之间,峰型较平。结论:实验结果表明,用本方法检测大批量人群标本的SNP结果符合遗传平衡定律,且操作简便,检测耗时短,结果特异且费用较为低廉,适合于进行大规模样品的SNP快速测定。     

ISSR标记在扁玉螺中的初步应用

以扁玉螺基因组DNA为材料,分析了Mg2 、dNTP、Taq DNA聚合酶、引物以及模板DNA浓度对ISSR-PcR扩增效果的影响,建立了一套适合扁玉螺的ISSR-PCR反应体系.该反应体系包括:2.0mmol/L的Mg2 ,0.25mmol/L的dNTP,0.8U的Taq DNA聚合酶,0.2μmol/L的ISSR引物和40ng模板DNA.利用优化后的反应体系,从30条ISSR引物中筛选出了13条扩增效果较好的引物,这为进一步利用ISSR标记研究扁玉螺的遗传多样性打下了基础.     

DNA分析仪的研制及性能考查

自行研制DNA分析仪,采用毛细管电泳分离DNA片段,激光诱导荧光技术进行检测。检测部分设计为共焦光路,488 nm的Ar＋激光器激发DNA片段上标记的荧光染料,发射的荧光信号由光电倍增管收集检测,激发光路和收集光路可三维调节。优化毛细管电泳-激光诱导荧光检测装置的设计和分析参数,实现了高分辨率、高灵敏度的DNA样品分析。实验结果表明,对于分析标准DNA样品pGEM-3Zf（＋）/HaeⅢMarkers和pBR322 DNA/HaeⅢMarker,标记的2种荧光染料分别为Thiazole Orange（TO）和SYBR GreenⅠ,本分析仪均获得了高信噪比的毛细管电泳图谱,较好地实现了单碱基分辨,检测灵敏度为2.4×10^-11mol/L。     

反刍动物精料补充饲料中牛羊源性成分-定性PCR方法

为防止疯牛病和痒病的传播，研究提供了一种利用CTAB从反刍动物精料补充饲料中提取动物基因组DNA的方法，并依据牛，羊基因组DNA的特异性序列片段，各设计1对引物，通过PCR扩增，实现了对反刍动物精料补充饲料中牛、羊源成分的检测。该方法简单、快捷，在饲料检测部门中有一定的推广价值。     

微生物检测用新一代DNA荧光探针检测仪

本文介绍了新一代DNA荧光探针检测仪,其原理为链置换扩增技术。该仪器在微生物检测中敏感、特异、快速,每日可检测500份以上样品,自动化程度高,操作简便,减少了污染。     

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.     

法医DNA样品自动化预处理平台布局设计及应用

目的:系统介绍自行设计研发的法医DNA样品自动化预处理平台的整体布局,并检测其提取效果,为该平台的使用建立依据。方法:基于磁珠提取法结合现有提取试剂盒的基本提取步骤,对该平台的分离、吸附、洗涤、洗脱等流程进行详细介绍,并针对血斑样本进行实际检测。结果:40个血斑样本在该平台上提取的DNA,其OD260/OD280均大于1.75,扩增产物在GA118-16A遗传分析仪上进行电泳,STR分型准确,位点完整,无等位基因丢失,图谱峰形尖锐清晰,并且信号较强。结论:法医DNA样品自动化预处理平台对整体布局及运动路径进行合理设计,从而加快了DNA提取速度,提高DNA提取纯度,可满足法庭科学DNA提取的应用要求。     

用于研究细胞入侵的基质降解实验

在细胞正常发育、发炎和肿瘤转移研究中有一个关键步骤,就是通过多层的细胞外基质入侵细胞.在传统的研究方法中,研究者们已经发展了自己的检测方法,就是利用荧光标记的凝胶来研究基质降解,其中伴随着细胞的入侵.这篇文章探索了研究细胞入侵的新方法,能得到比传统方法更一致的结果.研究者们利用这些方法来研究降解时间过程,扫描在降解过程中潜在的活化剂或者抑制剂,探索细胞入侵的通道.     

重压法干法制粒工艺及设备应用研究

应用重压法干法制粒工艺和设备对多潘立酮分散片、乙酰螺旋霉素片、盐酸氟桂利嗪胶囊、右旋布洛芬片和女金胶囊进行了制粒,比较了制剂重量差异、含量及溶出度,并对现行制粒工艺进行生产经济性分析。结果：各品种重压法干法制粒成品重量差异、含量及溶出度符合要求,生产成本较湿法制粒降低60％以上。结论：重压法干法制粒工艺在固体制剂成型处方进行适当调整的前提下对湿法制粒工艺具有质量和经济上的替代性。     

[智能感知]一种基于形态学特征的车道线识别方法

The existing lane detection algorithm mainly used edge information to extract lane features, and the algorithm that generated feature points through the contrast of adjacent pixels was easily affected by many external factors, and the detection results were easily disturbed, thus a new feature extraction algorithm was proposed. This algorithm improved the robustness of the algorithm in complex situations by calculating the tensor rotation of the gray structure in the region and selecting the largest changing trend pixel as the feature point. A new lane line feature extraction algorithm was used to extract feature points in interest areas, then feature points were selected, and Hough transform was used to fit them. After the lane lines were obtained , the dashed line and the solid line were distinguished by the coordinate variances of the feature points. The algorithm was tested by driving pictures of about 15 500 frames in different time periods. The results show that the detection method may detect the lane line well under various environments, the correct rate under the sunny conditions is as 99.18%, the correct rate under the rainy conditions is as 97.19%, and the correct rate under the worn pavement conditions is as 94.72%,the correct rate under the night conditions is as 97.62%.     

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct.

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100% identical to a rat DNA sequence (Accession No. NW_047400) downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.     

甲胎蛋白纯品的同位素稀释质谱法测定

建立了基于特征肽段的甲胎蛋白的液相色谱-同位素稀释串联质谱检测方法。选取3条同位素标记的甲胎蛋白特征肽段作为内标,准确称其质量后与酶切后的甲胎蛋白样品定量混合,采取Phenomenex Kinetex 2.6 μm C18色谱柱分离,电喷雾三重四极杆串联质谱多反应监测模式（MRM）测定,并对最优酶切条件、酶切效率以及定值结果的不确定度进行了考察和评定,得到的甲胎蛋白标准物质的最终测量结果为（0.329±0.016）mg/g。     

Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.     

粗蛋白质测定仪测奶制品蛋白质比较分析

目的：预期掌握目前部分大理产奶制品中蛋白质含量以及非蛋白质氮的含量情况,并与外包装指示含量进行比较与奶制品是否一致。方法：粗蛋白测定法,根据微量凯氏定氮法的原理,测定奶制品中总凝固沉淀物重量,及奶制品中非沉淀物经三氯乙酸处理后取沉淀再采用沉淀重量法测定蛋白质含量并与国家规定标准（简称＂国标＂）进行比较。结果：将调味牛奶、酸牛奶、纯牛奶、奶粉中总含氮物质与真蛋白质的含量进行比较,差异没有统计学意义,蛋白质含量与外包装上标注的蛋白质含量接近一致,蛋白质含量均达到国标以上。结论：目前部分大理产调味牛奶、酸牛奶、纯牛奶、奶粉中不含有非食用氮物质,蛋白质含量与外包装上标注的蛋白质含量接近一致,所测量的部分大理产奶制品的蛋白质含量符合国家奶制品蛋白质含量标准。