毛细管气相色谱法测定白芍中有机氯农药的含量

在建立白芍中七氯、环氧七氯及滴滴涕异构体含量的气相色谱分析方法中,样品以石油醚加丙酮在索氏提取器中提取,提取液以浓硫酸净化;采用DB-5(30 m×0.25 mm×0.25μm)弹性石英毛细管柱分离样品;以GC-ECD检测农药七氯、环氧七氯及滴滴涕的含量(色谱条件:载气为N2(99.999%,1.5 mL/min);分流比为2.3∶1;进样器温度为200℃,检测器测试为330℃,柱升温程序是从100℃(保持0.5 min)开始以15℃/min升至200℃保持1.0 min),再以4℃/min升至240℃(保持7 min);方法的加标平均回收率为96.27%~100.3%,精密度为2.0%~6.9%。     

毛细管电泳芯片制备工艺研究

叙述了采用SU-8和PDMS制作毛细管电泳芯片的一种新工艺、新方法,对SU-8的四个工艺参数：前烘温度与时间、后烘温度与时间、曝光时间以及显影时间进行了工艺优化,结果表明使用该方法加工工艺简单,芯片重复性好,成本低,适用于采用激光诱导荧光检测法进行检测,并在制备的毛细管电泳芯片上实现了不同长度DNA片段的快速有效分离．     

毛细管气相色谱法测定土壤和桔梗中七氯及滴滴涕的含量

建立了土壤和桔梗中七氯及滴滴涕异构体含量的气相色谱分析方法.样品以石油醚+丙酮在索氏提取器中提取,提取液以浓硫酸净化.采用DB-5弹性石英毛细管柱分离样品,GC-ECD检测农药七氯、滴滴涕的含量.方法的线性范围为2.18×10-13～5.31×10-11g;敏感度为3.4×10-12～6.8×10-12g/mL;加样平均回收率为96.27%～100.3%(RSD为3.0%～6.5%).     

茄子雄性不育株和可育株的胞质DNA和核DNA差异分析

以茄子(Solanum melongenaL)雄性不育株“正兴1号”(S)和可育株(F)的总DNA为模板,对60个随机引物进行了筛选,找到5个其RAPD(Random amplified polymorphic DNA,RAPD)扩增产物在茄子雄性不育系和对照材料间存在稳定差异的引物．将该5个引物同时扩增总DNA、核DNA和线粒体DNA(mitochondrial DNA,mtDNA)．以总DNA为模板时得到多态性片段9个,以核DNA为模板时得到5个,以mtDNA为模板时得到9个．以总DNA为模板时得到的9个扩增片段中,有5个在总DNA和mtDNA中同时出现,而在核中没有出现,即认为来自mtDNA,这5个片段中,有4个来自可育株,1个来自不育株,说明不育株和可育株在mtDNA上存在差异;有4个片段同时出现在以总DNA和核DNA为模板的扩增中,但在以mtDNA为模板的扩增中却没有出现,认为是来自核DNA,这4个片段中,有3个来自不育株,一个来自可育株,说明可育株和不育株在核DNA上也存在差异．结果初步表明：新发现的茄子雄性不育可能是核质相互作用所引起．     

Microdissection of chromosome 7B of common wheat and cloning of low-copy specific DNA sequences

The 7B chromosome of common wheat was microdissected from pollen mother cells of the 7B monosomic line of common wheat cv. Chinese Spring (CS). After proteinase K and DNA topoisomerase Ⅰ treatments, the isolated chromosomes were subjected to 1-3 rounds of DOP-PCR amplification, which produced continuous DNA fragments ranging from 150 to 700 bp. Ge-nomic Southern hybridization confirmed that the PCR products were originated from the wheat genome. Cloning of portion ( > 200 bp) of the 3rd round DOP-PCR products (50 μL) could generate about 20 000 recombinant clones. Characterization of 50 randomly chosen clones indicated that 21 clones produced discrete PCR products with the size of 240-600 bp. Dot-blot hybridization showed that among the 21 clones, 11 ( ～ 55%) were of low-copy nature while 10 ( ～ 45%) were repetitive. Southern hybridization with the complete set of the CS "nullisomic-tetrasomic (NT)" lines demonstrated that all the 6 low-copy clones were specific to either chromosome 7B or the 7th homoeologous group, whereas the 3 arbitrarily chosen repetitive clones were non-specific, disperse sequences.     

包囊游仆虫休眠细胞中大核和线粒体DNA的多态性

为了探索纤毛虫休眠细胞中两套遗传系统的作用特征,对包囊游仆虫（Euplotes encysticus）休眠细胞与营养细胞大核DNA和线粒体DNA进行了RAPD比较.结果显示,在所选用的35条随机引物中,包囊游仆虫大核DNA共扩增出220条片段,其中以休眠细胞大核DNA为模板扩增出18条特有片段,以营养细胞大核DNA为模板扩增出44条特有片段,两者存在28%的差异.在所选用的32条随机引物中,线粒体DNA共扩增出154条片段,其中以休眠细胞线粒体DNA为模板扩增出19条特有片段,以营养细胞线粒体DNA为模板扩增出25条特有片段,两者有29%的差异.这些结果表明,包囊游仆虫休眠细胞与营养细胞的大核DNA结构存着一定的差异;两者的线粒体DNA结构也存在差异.因此,包囊游仆虫在休眠细胞形成过程中,大核DNA、线粒体DNA结构可能都发生了一定的变化,并且这些变化可能与休眠细胞形成过程中的形态结构和代谢活动等剧烈变化以及休眠状态下的生理生化变化密切相关.所得结果为揭示纤毛虫细胞结构的分化与细胞遗传物质的作用关系提供了基础资料.     

电化学诱导铜(II)-磺基水杨酸配合物对DNA的切割

研究了在铜(Ⅱ)-磺基水杨酸配合物存在情况下的电化学诱导DNA的切割.铜(Ⅱ)-磺基水杨酸配合物在控制的-0.4V电位下,Cu(ssal)2 2作为媒介通过电化学反应产生活性氧自由基O2*,氧自由基可以切割DNA.被切割的DNA片断使用高效液相色谱可进行分离.实验结果表明,电化学诱导铜(Ⅱ)-磺基水杨酸配合物对DNA的切割方法简单,切割效果好.     

微卫星与生化位点法对BALB/c小鼠遗传检测比较分析

目的 应用微卫星DNA标记检测BALB/c小鼠的遗传质量,并与现行国家标准生化标记法进行比较,探讨其在近交系小鼠遗传监测中的应用,为建立微卫星DNA检测方法奠定基础。方法 采用20个微卫星基因位点和毛细管电泳技术对BALB/c小鼠进行遗传质量检测,同时采用现行国家标准生化标记法进行检测比较分析。结果 各样品20个微卫星基因位点DNA片段大小相同,没有新的等位基因出现;生化标记检测结果亦显示Akp1等14个生化标记基因均为纯合,个体间生化标记表型均一致,根据国家标准符合品系特征。微卫星DNA标记检测结果与生化标记检测结果一致。结论 微卫星DNA标记可准确可靠、方便快捷地检测近交系小鼠遗传质量,本研究所选的20个微卫星基因位点可用于BALB/c小鼠遗传质量监测。     

EcoRI核酸限制性内测酶切割Ad_5-DNA的动力学研究

本文用荧光扫描的技术研究了腺病毒5一型(Ad_5)DNA被EcoRI内切酶切割的动力学。这种技术能够定量地测定在琼脂糖凝胶上经溴乙锭染色的分离DNA片段的荧光强度,而且能根据各片段的荧光强度对切割时间的依赖关系,求得相应切割位点的切割速度。我们发现同一DNA分子上各切割位点的切割速度有着很大的差异,而且总的切割速度和酶—底物复合物的解离速度对酶浓度和温度有着明显的依赖性。切点两侧的碱基成分d(G-C)/d(A-T)比率高,则要求较多的活化能。     

Sm(Ⅲ)-HP配合物与DNA的作用机制

 应用紫外-可见光谱法和荧光光谱法并以中性红(NR)作光谱探针研究了Sm(Ⅲ)HP配合物与DNA的作用机制。研究表明,血卟啉(HP)与Sm(Ⅲ)的配合比n_Sm(Ⅲ)∶n_HP = 1∶ 1,Sm(Ⅲ)HP配合物与DNA的结合比n_Sm(Ⅲ)HP ∶ n_DNA = 1∶ 1,Sm(Ⅲ)HP配合物与DNA的结合常数K^θ_30℃ = 1.67×10⁴  L/mol。该过程为熵所驱动,其Δ _r H_m为4.86×10⁴ J/mol,Δ_r S_m为195.61 J/(mol·K),30 ℃时Δ_r G_m为－1.06×10⁴ J/mol。Scatchard法和探针法研究表明,Sm(Ⅲ)HP与DNA之间的结合方式主要是沟区作用。     

毛细管区带电泳法测定白藜芦醇与白藜芦醇苷

建立了以苄基三甲基碘化铵为内标测定白藜芦醇和白藜芦醇苷的毛细管区带电泳方法,在优化的分离条件下,两者在7 min内可基线分离,检出限均为1.25×10-4g/L,线性动态范围分别是4.88×10-4g/L~0.31 g/L和5.37×10-4g/L~0.34 g/L,样品的加标回收率为95.44%和97.75%.     

GAGE-1 DNA肿瘤疫苗的构建及其抗肿瘤治疗效果的试验研究

目的 观察G antigen 1 (GAGE-1)核酸疫苗pcDNA3.1+/GAGE-1免疫小鼠后,对表达GAGE-1抗原的B-16/GAGE-1肿瘤细胞的保护作用.方法 将C57 BL/6小鼠随机分为4组,与0、2、4周分别接种pcDNA3.1＋/GAGE-1（实验组1）、pcDNA3.1＋/GAGE-1/白介素2（实验组2）、pcDNA3.1＋（对照组1）,pcDNA3.1＋/白介素2（对照组2）各三次.末次免疫后10d小鼠用于肿瘤细胞攻击试验：分别于左背部、右背部皮下种植B16肿瘤细胞、B16/GAGE-1肿瘤细胞.种植肿瘤细胞（荷瘤）后观察成瘤时间、肿瘤大小和荷瘤后小鼠的生存时间及生存率. 结果： pcDNA3.1+/GAGE-1/IL-2质粒免疫的小鼠在种植B16/GAGE-1、B16/pcDNA3.1+后,发现小鼠成瘤时间明显延迟,成瘤减小,生存期明显延长.结论:pcDNA3.1+/GAGE-1/IL-2 DNA 疫苗在体内能诱导出显著的GAGE-1特异性肿瘤免疫应答,且能抑制体内已经存在的少量肿瘤细胞的成瘤     

不同球孢白僵菌菌株DNA的RAPD分析

采用RAPD技术分析了16个球孢白僵菌菌株的遗传分化。由160个随机引物中筛选出27个引物对各菌株进行了PCR扩增。结果表明从16个 孢白僵菌菌株中共获301个RAPD标记,其中多态性标记288个,占95．7％,表明菌株间具有较丰富的遗传多态性,聚类分析把16个球孢白僵菌菌株分为三大类型（1）Blh,,Bgd和Bpy;(3)By7,F-263,RECBb01,B12和B13;（3）B7,B11,Bxs,B6,By2,Bpc和Bzs。菌株DNA多态性与采集地之间有一定的相关性,但与寄主来源,孢子形态和对松墨天牛幼虫的毒力间未表现出明显的相关性。     

应用PCR—SSCP技术从显微切割的人染色体17q11—12探针池批量分离单拷贝片段的研究

将ＤＮＡ单链构象多态（ＳＳＣＰ）检测的原理，应用于“显微切割的人染色体１７ｑ１１—１２探针池”批量分离单拷贝片段，取得了很好效果。从筛选出的７４个次级单拷贝中有效地鉴定出３７个非同源的单拷贝片段。这比传统的仅限于长度比较而确认的非同源单拷贝片段（１２个）多出２５个．其中部分已经ＤＮＡ测序证实。结果提示：采用ＳＳＣＰ技术区分相似分子量单拷贝片段的同源性，可显著地提高从“显微切割探针池”分离和克隆非同源单拷贝片段的效率。本文还将一部分单拷贝片段作为探讨，与人基因组ＤＮＡ的限制性片段作Ｓｏｕｔｈｅｒｎ印迹杂交，结果均只显示单一杂交带，说明单拷贝ＤＮＡ片段的结论是可靠的。     

Comparative Studies on Preparation of Large Plant DNA Fragments by Pulse Field Gel Electrophoresis

The preparation of large plant DNA fragments is extremely important to the construction of large insert DNA libraries (YAC, BAC, PAC and TAC). Although several techniques have been developed in each step of large plant DNA fragments preparation, the whole processing remains complicated and difficult. Based on authors‘ research experience and the recent worldwide development in this field, the following aspects are discussed in this paper: techniques of plant high molecular weight (HMW) DNA purification by pre-electrophoresis, the optimal conditions for the partial digestion of the HMW DNA by HindⅢ, the isolation effects of of large plant DNA fragments (100-400 kb) with different parameters of pulse field gel electrophoresis (PFGE), and the recovery of large DNA fragments. Through comparative studies, the advantages and disadvantages of each technique are discussed and some recommendations are proposed for preparing high quality large plant DNA fragments. The suggested techniques have been used in preparing the large DNA fragments of maize, rice, moss, laver, sea tangle and peach, and similar resuits are obtained among all the materials. This paper only reports the results using maize as material.     

GC/MS法测定杀虫剂甲拌磷中的杂质

应用毛细管气相色谱和质谱法对杀虫剂甲拌磷原油进行了很好的分离和分析,通过MSLS质谱数据检索系统对多数杂质进行了定性。讨论了甲拌磷及两种硫代磷酸酯的质谱特性,并通过质谱解析对主要碎片和裂解机理加以证实。     

Mechanism of oxidative damage to DNA by Fe-loaded MCM-41 irradiated with visible light

The mechanism of oxidative damage to deoxyribonucleic acid (DNA) by iron-containing mesoporous molecular sieves (MCM-41) irradiated with visible light was elucidated. Fe-loaded MCM-41 (Fe/MCM-41) was used as a photocatalyst and the damage to calf thymus DNA caused by hydrogen peroxide (H2O2) was studied. The damage and extent of oxidation of DNA were measured by high-performance liquid chromatography (HPLC) and intermediate products were detected by HPLC/electrospray ionization tandem mass spectrometry. Electron spin resonance was used to detect changes in reactive oxygen species and peroxidase catalytic spectrophotometry was used to determine the concentration of H2O2. The results indicated that Fe/MCM-41 efficiently activated H2O2 in solution at pH 4.0-8.0 under irradiation with visible light. The photocatalytic system degraded DNA most effectively at pH 5.0-6.0 but also operated at pH 8.0. At pH 4.2, the degree of DNA damage reached 25.65% after 5 h and the kinetic constant was 5.89×10 2 min 1. Damage to DNA was predominantly caused by hydroxyl radicals generated in the system. The mechanism of DNA damage is of potential concern to human health because it can occur in neutral solutions irradiated by visible light.     

大鼠肝再生中肝细胞基因表达与DNA裸露的相关性研究

为了解大鼠肝再生中肝细胞的基因表达与DNA裸露的相关性,分离大鼠肝细胞,提取裸露DNA,用SOLEXA方法对DNA测序,用BWA软件拼装测序片段,用IPA软件分析拼装的基因种类和作用,用Rat Genome230 2.0芯片检测基因的转录情况.研究表明,大鼠肝再生的12h(PH后12h),肝细胞的裸露DNA涉及16 912个基因,1 701个基因发生了有意义裸露量变化.其中,25个基因的裸露量上调,1 676个基因的裸露量下调.与Rat Genome 230 2.0芯片的检测的基因转录比对表明,肝细胞的AKR7A3,BMYC,CDC25B等26个基因的裸露量和表达量均下调,表明大鼠肝再生12h时,这些基因的表达可能受染色体结构调控.     

瓢虫干标本基因组DNA的提取及RAPD分析

实验对保存多年的瓢虫干标本进行了基因组 ＤＮＡ提取和 ＲＡＰＤ－ＰＣＲ扩增。结果显示 ＤＮＡ分子的提取与保存年代长短无直接关系;ＲＡＰＤ－ＰＣＲ增产物经琼脂糖凝胶电泳检测,分子量大小约为 １９８４ｂｐ～６３０ｂｐ;不同种瓢虫的 ＤＮＡ用同一引物,扩增片段是多态表现,同一种瓢虫的干标本保存时间虽不同,但扩增产物中均具有相同的ＤＮＡ片段。     

Interaction between poly（amidoamine） dendrimers and DNA studied by spectroscopic methods

The interaction between amino-terminated, and ethylenedtamine core poly（amtdoamine） （PAMAM） dendrimers and herring sperm DNA was investigated by various spectroscopic methods including UV spectroscopy, fluorescence spectroscopy, microscopic FTIR- and circular dichroism （CD-） spectroscopy. Ethidium bromide （EB） is used as a nucleic acid probe for this study. Experimental results show that PAMAM dendrimers can form stable complexes with DNA and the dendrimers bind to DNA sufficiently strong which cannot be displaced by EB, and we also found that the formation of the complexes can cause the conformation change of the DNA secondary structure. According to the Scatchard analysis, the association constant of PAMAM to DNA is calculated to be 2.53×10^4 mol/L^-1.