实时定量PCR鉴定转基因作物纯合体

以水稻内标准基因磷脂酶D基因(phospholipase D,PLD)和转基因水稻TT51-1特异性旁侧序列为检测靶标,对单株转基因水稻的种子播种后长出的单株进行了荧光定量PCR,以其内标准基因和旁侧序列Ct值的差值判断了植株的纯合体,并用标准曲线计算了转基因含量,证实了此法的可靠性,说明此法用于鉴定植株的纯合体简便准确.     

单壁碳纳米管对长片段聚合酶链式反应(PCR)增效机理的初步研究

为了开发新型基因扩增技术,采用两步PCR(two-step PCR)和rpsL保真度实验等方法,对单壁碳纳米管(single-wall carbon nanotube,SWCNT)在长片段PCR反应中的增效机理进行研究.结果表明:在长片段PCR反应中,单壁碳纳米管最适工作质量浓度为0.8,mg/mL;单壁碳纳米管可以在较短的延伸时间增加扩增产物产量,提高DNA聚合酶的反应效率;通过rpsL保真度实验显示,对照组PCR产物的错误率为5.32×10–6,而实验组的错误率仅为2.39×10–6.     

脉胞菌hH3v基因的初步研究及原核表达载体构建

根据植物表达载体pET52（b）多克隆位点和脉胞菌着丝粒结构相关蛋白CenH3的编码基因hH3v序列，设计引物，通过PCR扩增将基因两端加上KpnⅠ和SacⅠ酶切位点，经双酶切后将hH3v插入表达载体pET52（b）中，利用热激法转化大肠杆菌Top10感受态细胞，在选择培养基上挑取单菌落培养，经PCR检测，表明目的基N原核表达载体构建成功，为着丝粒结构进一步的研究提供了基础．     

Enhanced activity of yqhD oxidoreductase in synthesis of 1,3-propanediol by error-prone PCR

yqhD oxidoreductase was determined to be an NADP-dependent dehydrogenase, and was more active toward 3-HPA when compared to 1,3-propanediol oxidoreductase. To further improve enzyme activity towards 3-hydroxypropionaldehyde （3-HPA）, error-prone PCR was implemented to mutant yqhD gene. Two mutants, D99QN147H and Q202A with increased catalytic and affinity efficiency, were obtained after one round of error-prone polymerase chain reaction. And the catalytic efficiency of the mutant D99QN147H was up to 4-fold greater than the wild enzyme （0.0375 min^-1 mM^-1 vs. 0.0078 min^-1 mM^-1）. The recombined strain containing pET28yqhD D99QN147H yielded 28 g L^-1 of 1,3-propanediol in the fed-batch LB cultures （1 L volume） with an initial 3-HPA concentration of 40 g L^-1, which was higher than the 21 and 17 g L^-1 of 1,3-propanediol from the mutant Q202A and the wild-type, respectively. Except for propionaldehyde, the optimal mutant D99QN147H also exhibited higher activity on a range of substituted aldehydes than the wildtype.     

原位多聚酶链式反应（In Situ PCR）技术的应用和发展

原位ＰＣＲ是一项新的分析子技术，它结合了杂交技术和ＰＣＲ技术的优点，具有高度的敏感性，特异性，并能进行精确的定位，在研究工作和临床诊断上有广阔的应用前景。     

苯并芘诱发的人支气管癌细胞系p53基因突变的研究

ＢＴ癌系是用苯并芘称在移植到裸鼠皮下的人胎儿支气管内诱发的肿瘤，为了深入了解苯并芘的致癌机理，我们对该癌系中ｐ５３基因的改变进行了系统的研究。结果表明，ＢＴ细胞核内ｐ５３蛋白异常高表达；ＰＣＲ－ＳＳＣＰ检测到第７外显子有异常改变；Ｄ 列分析证实第２４８密码子发生了ＣＧＧ→ＣＴＴ的颠换，所编码的氨基酸由精氨酸变为亮氨到。本实验提示该害变可能是七产芘引起癌变的重要分子基础。     

几种动物RAPD指纹图谱反应条件的分析

用随机排列碱基顺序的寡核苷酸引物，对蚕、鱼、蛇、家猪等动物的基因组ＤＮＡ扩增，获得了这些动物的随机扩增多态ＤＮＡ指纹图谱，对影响扩增结果的几个因素进行了分析，对动物随机扩增多态ＤＮＡ反应的适宜条件进行了讨论。     

用加端PCR技术改造h－IGF－1cDNA

用ＤＮＡ合成仪合成了分别带有ＰｓｔＩ位点和ＳａｌＩ位点及终止密码子的２个用于扩增ｈＩＧＦ－１ｃＤＮＡ的ＰＣＲ引物．利用合成的引物，７００ｂｐ长的ｈＩＧＦ－１ｃＤＮＡ模板和Ｔａｑ聚合酶进行ＰＣＲ扩增．扩增产物经电泳鉴定后克隆进Ｍ１３ｍｐ１８载体，进行核苷酸序列分析．结果显示：ＰＣＲ产物含已发表的ｈＩＧＦ－１成熟蛋白的编码序列和５＇端的ＰＳｔＩ位点及３＇端的ＳａｉＩ位点及终止密码ＴＡＧ．用加端ＰＣＲ技术成功地扩增和改造了ｈＩＧＦ－１的编码序列．     

Studies inin situ PCR detected by the BrdU antibody technique

Using the BrdU antibody technique followed by an immuno-chemical staining (BAT), the amplification of DNA fragments specific to human Y chromosome on cell specimen slides was efficiently detected. Whether direct BrdU incorporation into PCR products orin situ hybridization with PCR products on slides, the amplified target DNA fragments of specimen were visualized by BAT under the microscope. The availability of BAT and differences in the sensitivity and efficiency between BAT and dig-11-dUTP labeling in cellin situ PCR were discussed. Zhang Xiyuan: born in Oct. 1935, Professor, Current research interest in Cellular and Molecular Biology Supported by the National Natural Science Foundation of China     

胚胎种植前遗传学诊断研究进展

目的:综述胚胎种植前遗传学诊断(PGD)研究最新进展,以指导临床实践。方法:光盘检索相关文献并进行综合分析。结果:PGD是进行优生优育的重要方法,主要采用聚合酶链反应技术(PCR)与荧光原位杂交技术(FISH)。结论:PGD具有广阔的应用前景。