模糊PID复合控制算法改进及应用

针对常规模糊PID复合控制算法的不足,在分析了其控制特性后,提出了相应的改进算法.采用基于梯形隶属函数的模糊切换算法以及基于变论域和仿人智能思想的量化因子和比例因子的在线自调整算法实现了自适应模糊PID复合控制,并将此改进算法用于PCR芯片智能温度控制系统中,使控制系统获得了良好的动态特性和稳态性能、较强的鲁棒性及适应性.实验结果表明,其控制品质远优于常规模糊PID控制和基本模糊控制.     

荧光定量PCR技术及其在动物病毒病定量检测中的应用

荧光定量PCR技术是一种核酸定量技术,该技术在PCR反应体系中加入荧光基团,利用荧光信号累积实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法.该技术具有特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等特点,因此在动物病毒病的检测中,荧光定量PCR技术不但能定量检测病原体感染的强弱和在机体内的分布,还具有灵敏、快速、省力的特点.     

一种新的基因克隆方法SLIC的可靠性分析

Li & Elledge 最近报道了一种新的不依赖于基因序列和连接反应的高效基因克隆方法(Sequence and Ligation Independent Cloning,SLIC).这种方法以同源重组为基本原理,利用T4 DNA聚合酶在插入片段以及载体的末端产生单链突出末端,通过同源序列在体外完成交换重组,达到基因克隆的目的.本研究通过大肠杆菌蓝白菌落筛选和对重组子的酶切分析,证明SLIC是否能够使DNA片段在设计的位点处发生精确的重组,实验证实,SLIC的重组是非常可靠的.     

RNA干涉

RNA干涉是指双链RNA引发的同源mRNA的降解过程,由于其多发生于转录水平,因此又称为转录后基因沉默。RNA干涉现象是在线虫中首次发现的,通过几年的研究发现它广泛存在于生物体中(包括单细胞生物、后生动物和哺乳动物)。本文对RNA干涉现象的发现、作用机制、相关基因等作了简单的论述。     

应用FQ-PCR检测孕妇外周血中胎儿细胞含量

合成一对染色体单拷贝基因GAPDH特异的引物和一对Y染色体单拷贝基因SRY特异的引物,并合成两条特异性TaqMan探针,加等量的DNA到含有上述两个引物探针的PCR反应体系中,行荧光定量PCR,获得两种基因的定量值,通过这两种基因含量的比值,计算孕妇外周血中胎儿细胞含量.在30 μL FQ-PCR反应体系中,加入最少约0.05 ng 正常男性基因组DNA能够稳定地获得扩增曲线;在60例临床孕妇外周血标本DNA 的DCFQPCR扩增中有35例出现SRY扩增阳性,阳性率为58.3%;在这些标本中,GAPDH 基因Ct值范围在20~22之间;SRY 基因Ct值范围在33~40;经计算,胎儿细胞含量范围在1/10.4~1/10.6.     

DNA聚合与RNA转录联用分子机器的改进

建立了一个集成DNA聚合和RNA转录过程,能够重复产生RNA适体片段,并结合孔雀石绿产生荧光信号的分子机器,并探索了此分子机器在检测DNA方面的应用.转录产生的大量孔雀石绿适体序列被释放到溶液中,并可以与孔雀石绿结合产生荧光,实现信号的放大.85μL体系中的聚合转录的最适条件为：DNA聚合酶10 IU（0.118 IU·μL-1）,RNA聚合酶60 IU（0.706 IU·μL-1）,反应时间为3h.在上述条件下,荧光信号随着引发DNA用量的增加而增大.     

Apoptosis Induced by High Concentrations of Nicotinamide in Tobacco Suspension Cells

As an inhibitor of poly(ADP-ribose) polymerase (PARP), nicotinamide has a restraining effect on apoptosis at certain low concentrations. In our present study, apoptosis induced by high concentrations of nicotinamide was observed in tobacco suspension cells. When cells were preincubated with 250 mmol/L nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180-200 bp，the condensation and peripheral distribution of nuclear chromatin, and a positive reaction to the TUNEL assay. At the same time, the degradation of PARP and the reduction in the potential of the inner membrane of mitochondria appeared in apoptotic cells induced by high concentrations of nicotinamide. This result indicates that apoptosis induced by high concentrations of nicotinamide is associated with caspase-3-1ike activity and with the opening of mitochondrial permeability pores. These results partially support the hypothesis that high concentrations of PARP inhibitor could force cells to enter an apoptotic pathway by delay of DNA repair in replicating cells.     

实时定量聚合酶链反应的研究及应用(综述)

实时定量聚合酶链反应（real-time quantitative PCR,RQ-PCR）是综合PCR、分子杂交和酶动力学的一种新技术，它根据荧光共振能量转移（fluorescence resonance energy translation,FRET）的原理进行。目前已经成功地开发出TayManTM、molecular beacon probe、amplifluor technologies、LightCycler和single-labeled probe几种相关的技术，并广泛地应用于病毒学、遗传性疾病和肿瘤等方面的诊断检测。实验研究表明：RQ－RCR作为一种快速、准确的核酸检测方法，在临床及生物学研究方面均具有相当的实用价值。     

T7启动子促发下的人尿激酶原在大肠杆菌中的高效表达

将人工全化学合成的尿激酶原（ｐｒｏ－ｕｒｏｋｉｎａｓｅ）ｃＤＮＡ克隆到表达载体ｐＥＴ３ｄ中，转化大肠杆菌ＢＬ２１（ＤＥ３）ＬｙｓＳ，经ＩＰＴＧ诱导，获得了占菌体总蛋白１８％的高表达。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ鉴定，表达产物主要为单链尿激酶，并且在菌体内基本以无活性的包涵体形式存在。经体外变复性，从ＩＬ培养基中可获得３０００００单位的活性尿激酶原。     

Assaying poly(ADP-ribose) polymerase activity in plants by polarographic method

A new method has been developed to assay poly(ADP-ribose) polymerase (PARP) activity in plant tissues through determining the content of nicotinamide (NIC) produced by enzymatic reaction by linear sweeping polarographic method. The detection limit of NIC was 0.03μmol/L, the calibration graph was linear up to 5 Mmol/L ( r = 0.999). The recoveries were approximately in the range of 92% to 98% and the relative standard deviations were less than 6.6% . Moreover, NAD+ and other interference existing in the mixture after enzymatic reaction had been removed by simple pretreatment, thus PARP assays were not interfered. A rapid, simple, sensitive and reliable nonisotopic method is reported to assay PARP activity in plant tissues . The results show that the KmNAD+ value of PARP in maize ( Zea mays L.) seedlings is 59 and the optimum pH for PARP activity is 8.5. Moreover, physiological conditions affect PARP activity in plant tissues, which has not been reported previously. When tobacco ( Nico-tiana tobacum) suspension cells were stressed by NaCI at low concentrations (100, 200 mmol/ L), the PARP activity increased significantly; when the cells were stressed at high concentrations (400, 1 000 mmol/L), it decreased to or even below the control level. PARP activity in etiolated maize seedlings was higher than that in light-grown seedlings.