双加氧酶α亚基保守序列克隆及探针制备

制备地高辛标记的环羟基化双加氧酶的α亚基保守序列探针。利用PCR法成功地克隆了环羟基化双加氧酶的α亚基保守序列310 bp片段,并对其进行测序。利用PCR法制备地高辛标记探针,对新标记的探针进行检测,结果显示其标记效率在0.1 pg/μL;敏感性检测表明,对同源DNA的检出限量为100 pg;对提取的副溶血性孤菌质粒DNA、短小芽胞杆菌总DNA杂交均呈阴性,说明该探针具有较强的特异性。     

用地高辛标记Y染色体特异探针检测人的性别

采用了一种新的DNA探针非同位素标记方法,即地高辛配基标记人Y染色体特异DNA探针,成功地用于人基因组中男性特异DNA的检测。同时,将此种方法与生物素标记探针方法作了比较。结果表明：地高辛配基标记探针方法优于生物素标记。     

两种末端标记的寡聚核苷酸探针的比较研究

本文利用[α-³²P]-dATP 和 [γ-³²P]-ATP分别对寡聚核苷酸的3’-末端及5’-末端进行标记,研究这两种标记方法的技术途径,并结合斑点杂交,比较这两种末端标记的探针的灵敏度及特异性。结果表明:[α-³²P]-dATP虽可在寡聚核苷酸的3’-OH末端标上多个[³²P]-dAMP分子,但所得标记探针的杂交灵敏度及杂交体的稳定性均不及[γ-³²P]-ATP标记5’-末端制备的寡聚核苷酸探针。因此,当实验需要极高灵敏度的寡聚核苷酸探针时,以选择[γ-³²P]-ATP标记其5’-末端为宜。     

对大豆根边缘细胞程序性死亡诱导的生理生态作用

设置不同的Al ³⁺浓度（0、25、50、100、200、400 μmol·L^-1）和培养时间 （12、24 h）,研究了边缘细胞活性和大豆（Glycine max）根中 过氧化氢酶（CAT）、过氧化物酶POD）、超氧化物歧化酶SOD）随Al ³⁺浓度及处理时间变化的规律,并通过Hoec hst333 42-PI双重荧光染色、 梯状DNA（即DNA ladder）分析和末端脱氧核糖核酸转移酶介导的dUTP切口末端标记（即TUNEL原位标记）检测,研究了Al ³⁺对大豆根边缘细胞 程序性死亡诱导的生理生态作用。结果表明,Al ³⁺胁迫能诱导边缘细胞的死亡,随着Al ³⁺浓度的升高和处理时间的延长,细胞死亡率增加。通 过Hoechst33342-PI双重荧光染色、DNA ladder分析和TUNEL原位标记,检测到Al ³⁺胁迫下发生程序性死亡的边缘细胞。其表现为：在 400μmol·L^-1 Al ³⁺诱导大豆根24 h时, 核酸电泳显示细胞DNA发生特异性降解并形成阶梯状电泳条带(DNA ladder),用TUNEL原位标记检测200 和400μmol·L^-1 Al ³⁺处理12 h后的大豆根 边缘细胞,发现DNA的3′-OH端被原位特异标记,二氨基联苯胺（DAB）显色后,细胞核为阳性或强 阳性。同时,高浓度Al ³⁺ （＞100μmol·L^-1）处理下,CAT、POD和S OD活性均有不同程度的下降,CAT和SOD的活性也随处理时间的延长而降低 。说明在Al ³⁺胁迫下边缘细胞的死亡可能是一种程序性死亡形式,高浓度Al ³⁺胁迫下,通过诱导活性氧在细胞体内的产生和累积而导致细胞凋 亡,此过程是其对逆境胁迫所作出的生理生态防御性应答方式之一。     

Al3+对大豆根边缘细胞程序性死亡诱导的生理生态作用

 设置不同的Al ³⁺浓度（0、25、50、100、200、400 μmol·L^-1）和培养时间 （12、24 h）,研究了边缘细胞活性和大豆（Glycine max）根中 过氧化氢酶（CAT）、过氧化物酶POD）、超氧化物歧化酶SOD）随Al ³⁺浓度及处理时间变化的规律,并通过Hoec hst333 42-PI双重荧光染色、 梯状DNA（即DNA ladder）分析和末端脱氧核糖核酸转移酶介导的dUTP切口末端标记（即TUNEL原位标记）检测,研究了Al ³⁺对大豆根边缘细胞 程序性死亡诱导的生理生态作用。结果表明,Al ³⁺胁迫能诱导边缘细胞的死亡,随着Al ³⁺浓度的升高和处理时间的延长,细胞死亡率增加。通 过Hoechst33342-PI双重荧光染色、DNA ladder分析和TUNEL原位标记,检测到Al ³⁺胁迫下发生程序性死亡的边缘细胞。其表现为：在 400μmol·L^-1 Al ³⁺诱导大豆根24 h时, 核酸电泳显示细胞DNA发生特异性降解并形成阶梯状电泳条带(DNA ladder),用TUNEL原位标记检测200 和400μmol·L^-1 Al ³⁺处理12 h后的大豆根 边缘细胞,发现DNA的3′-OH端被原位特异标记,二氨基联苯胺（DAB）显色后,细胞核为阳性或强 阳性。同时,高浓度Al ³⁺ （＞100μmol·L^-1）处理下,CAT、POD和S OD活性均有不同程度的下降,CAT和SOD的活性也随处理时间的延长而降低 。说明在Al ³⁺胁迫下边缘细胞的死亡可能是一种程序性死亡形式,高浓度Al ³⁺胁迫下,通过诱导活性氧在细胞体内的产生和累积而导致细胞凋 亡,此过程是其对逆境胁迫所作出的生理生态防御性应答方式之一。     

用Digoxigenin标记DNA探针作基因转移山羊的整合检测

对采用外源基因原核显微注射法生产的基因转移山羊,用一种新的DNA非同位素标记法即地高辛配基标记DNA探针作外源基因整合检测.结果表明:地高辛标记核酸探针能检测出基因转移动物基因组中整合的外源基因.     

地高辛标记DNA探针杂交法检测Vero细胞DNA残留量的适用性验证及应用

目的对地高辛标记DNA探针杂交法检测人用狂犬病疫苗(Vero细胞)DNA残留量进行适用性验证及应用。方法对地高辛标记DNA探针杂交法检测人用狂犬病疫苗(Vero细胞)DNA残留量进行特异性、灵敏度及稳定性验证,并应用该方法检测3批人用狂犬病疫苗(Vero细胞)的DNA残留量。结果地高辛标记探针的标记效率为0.1 pg。验证结果显示探针与非同源DNA无杂交;最低检测限度为1 pg;探针在-20℃放置7个月后,检测灵敏度仍可达到1 pg;3批人用狂犬病疫苗(Vero细胞)中残留DNA含量均符合《中国药典》规定质量控制标准。结论地高辛标记DNA探针杂交法特异性、灵敏度好,结果稳定,适用于人用狂犬病疫苗(Vero细胞)中Vero细胞DNA残留量的检测及疫苗生产过程和其成品的质量控制,对其他以Vero细胞为基质的病毒性疫苗质量控制具有借鉴意义。     

125Ⅰ标记乙型肝炎病毒DNA探针的制备及应用

本文报道用国产¹²⁵I-碘化钠标记乙型肝炎病毒DNA制备探针,可得到较高比度的产物,一般稳定在10⁷—10⁸cpm/μg DNA。用该探针对不标记的乙型肝炎病毒DNA进行点分子杂交。可检测到5pg左右DNA。对17例血液标本进行点分子杂交,结果与32p-标记乙型肝炎病毒DNA探针杂交结果基本一致。     

PCR法制备地高辛标记探针改良PCR-Select Differential Screening试剂盒

目的:探讨采用地高辛(digoxin,DIG)标记探针替代PCR-Select differential screening试剂盒中同位素标记探针的方法.方法:抑制性消减杂交(suppression subtractive hybridization,SSH)所分离的差异表达序列转移至硝酸纤维素膜,以PCR法掺入DIG-11-dUTP制备探针,按DIG标记探针常规操作进行杂交显色,杂交阳性结果采用reverseNorthern blot再度杂交验证.结果:应用DIG标记探针改良PCR-Select Differential Screening试剂盒所得到的阳性结果经reverse Northern blot验证符合率达到90%.结论:DIG标记探针改良PCR-Select differential screening试剂盒在避免了放射污染同时具备很高的特异性,完全可以替代同位素标记探针.     

党参基因组DNA提取、ISSR-PCR反应体系优化及引物筛选

对党参基因组DNA提取方法优化、ISSR体系形成及引物筛选三方面进行探讨,为研究党参居群遗传多样性及药材DNA鉴定奠定基础。经比较改良CTAB法、改进的高盐SDS法和试剂盒三种常用DNA提取方法,发现改良CTAB法效果最佳;利用优化设计并结合有关文献优化ISSR反应体系,最优反应体系为：50 μL总反应体积中含约20 ng DNA模板,1.25 U Taq DNA聚合酶,2.25 mmol·L^-1 Mg²⁺,200 μmol·L^-1 dNTP,0.50 μmol·L^-1引物。以此体系为基础进行引物筛选,在100条ISSR引物中筛选出13条扩增条带清晰、多态性较高、重复性好的引物。     

Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis

Improvements to the sensitivity, speed, and reproducibility of digoxigenin (DIG)-labeled probes and chemiluminescent substrates makes these compounds increasingly popular to detect nucleic acids. High sensitivity and low background are essential in Southern blot analysis, particularly with plant DNA. This article describes a nonradioactive system to detect single-copy genes in transgenic plants. Labeling using the polymerase chain reaction (PCR) was employed to produce highly sensitive and reusable DIG-labeled probes. The background was reduced by immobilizing the DNA onto nylon filters by alkaline transfer and by minimized gel handling; the signal-to-noise ratio was improved by modification of the detection procedure.     

Comparison of commercial probe labeling kits for microarray: towards quality assurance and consistency of reactions

Microarray technology is readily available to scientists interested in gene expression. Commensurate with this availability is the growing market in accessory products offering convenience but potentially variable performance. Here we evaluate seven commercial kits for probe labeling against a human apoptosis oligonucleotide array. All kits were found to label probes successfully using the manufacturers' instructions. The Stratagene Fairplay Microarray Labeling Kit was the most sensitive, with an overall call rate of 74% and the lowest rate of indeterminant calls for the HEK and HepG2 cell lines. The Invitrogen SuperScript Indirect cDNA Labeling System showed the most reproducible gene expression pattern and the least technical variation, both in terms of signal strength and between replicates on each array. The Promega Pronto! Plus System showed the least dye bias however, a higher level of variation between replicates was observed. Pairwise comparisons revealed that the Promega Pronto! Plus System and Invitrogen SuperScript Indirect cDNA Labeling System had the most similarity in their patterns of gene expression. Results obtained suggest variability in the performance of commercial kits between different manufacturers. This study supports the need to conduct comparative evaluations of commercial microarray probe labeling kits and the need for validation prior to use.     

带有白花甜菜染色体片段的栽培甜菜易位系的筛选

本文采用斑点杂交方法,对近100%传递的甜菜单体附加系M14(2n=19)发生分离的后代反转系(2n=18)进行研究,采用随机引物法标记白花甜菜特异探针,对80株反转系进行杂交实验,结果筛选出19个易位系,这19个易位系有待于进一步研究,证明是否带有无融合生殖基因。     

DIG标记DNA探针检测noggin在爪蟾胚胎中的表达

整体原位杂交（Whole-mount in situ hybridization)用于基因表达定位和表达分布模式的研究,已经成为一种非常重要的手段。PCR方法进行探针标记,可以获得特异性高,片断大小可变的探针。采用DIG标记,检测已知基因noggin在爪蟾胚胎时空分布,所得结果与文献报道一致,表明PCR方法获得的DNA探针在一定的条件下可以用于爪蟾胚胎整体原位杂交。     

Improvement of the dot-blot-SNP technique for efficient and cost-effective genotyping

Although the dot-blot-SNP technique is a laborsaving, cost-effective method for SNP genotyping of a large number of plants, the synthesis of 5′-digoxigenin (DIG)-labeled oligonucleotides for use as probes is still costly. We developed two probe-labeling methods for this technique, one being digoxigenin labeling of oligonucleotides by PCR (PCR-DIG labeling) and the other being hybridization using a bridge probe and a 5′-DIG-labeled oligonucleotide (bridge hybridization). Bridge hybridization detected allele-specific signals under hybridization conditions similar to those for the 5′-DIG-labeled oligonucleotides and biotin-labeled oligonucleotides, while signals were detected only under a lower stringency condition by PCR-DIG labeling. As a method for genotyping using many markers at one time, two methods, i.e., PCR using mixed primer pairs and hybridization using mixed probes, were examined with successful results. Eighty-five SNP markers designed for genotyping of rice cultivars detected allele-specific signals, the genotyping results corresponding to the previously reported ones.     

Effect of particle size on labeling density for catalase in protein A-gold immunocytochemistry

Effect of particle size on labeling intensity in protein A-gold immunocytochemistry was studied. Catalase labeling of rat liver peroxisomes was used as a labeling model. Ultra-thin sections of Lowicryl K4M-embedded rat liver were stained for catalase with protein A-gold (pAg) probes. Five different sizes of colloidal gold probes, from 5 nm to 38 nm in diameter, were prepared. Labeling intensity decreased as the particle size of the pAg probes increased. The highest labeling was obtained by the 5-nm pAg probe and the lowest by the 38-nm pAg probe. Quantitative analysis also showed that labeling density was inversely proportional to the size of gold particles. The results suggest that the pAg probe with small gold particles has high sensitivity.     

通过荧光标记的凝胶阻滞技术分析Opaque2蛋白与ZmGRAS11启动子的结合位点

凝胶阻滞实验（electrophoretic mobility shift assay，EMSA）是研究蛋白质与核酸结合的一种关键实验技术。EMSA技术兴起以来，使用放射性同位素、生物素标记核酸探针的手段已经非常成熟，但这两种传统的标记技术分别具有放射性探针稳定性差和生物素检测步骤复杂等缺点。近年来，尽管荧光标记探针逐渐被应用于EMSA中，但是对于利用荧光标记探针的EMSA仍缺乏系统的报道。对荧光标记的EMSA技术流程进行了优化和系统总结；利用6-羧基荧光素（6-carboxy-fluoroscine，FAM）标记ZmGRAS11启动子探针，通过EMSA检测其与Opaque2蛋白的结合，明确了蛋白和探针的适宜比例为8∶1。对GCN4 motif序列碱基进行突变并利用EMSA分析Opaque2与ZmGRAS11启动子之间的结合位点，结果表明GCN4 motif的“TGAC”核心基序在ZmGRAS11启动子与Opaque2蛋白的结合中可能起到了关键作用。研究结果为进一步探究Opaque2-ZmGRAS11转录调控模块在玉米籽粒发育中的作用机理提供了数据支撑。     

Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe.

The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grünwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.     

Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe

Summary The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated. Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter. Cytocentrifuge preparations were made from the cell suspensions. Silver enhancement was performed on all preparations. Then they were counterstained with May-Grünwald Giemsa and examined in light microscopy. The immunostaining appeared as fine dark granules on the surface membrane of the cells. Labeling conditions were determined which gave a dense specific immunostaining and a low background. High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples. The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles. Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy. We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.