以寡聚核苷酸为探针对癌基因Ha-ras点突变的测定

<正> 基因中一个核苷酸的改变(点突变)是原癌基因活化的途径之一。核苷酸序列分析表明,我们从胃癌细胞系中克隆出的癌基因Ha-ras,就是由于其编码区第35位核苷酸从鸟嘌呤(G)变为胸腺嘧啶(T)而被激活的。测定基因的点突变除了序列分析方法之外,还有比较简单、经济的寡聚核苷酸探针分子杂交等方法。我们按照正常的原癌基因c-Ha-ras和胃癌     

Fluorescent-Labeled Oligonucleotide Probes with Non-Nucleotide Linker: Detection of Hybrid Formation by Fluorescence Anisotropy

Abstract

Fluorescein labeled oligonucleotide probes with non-nucleotide linker have been synthesized and used to monitor hybrid formation to detect DNA sequences in solution. Fluorescence anisotropy, r, was adopted as an index to monitor triple helix formation and the behaviour of F-Probe in solution. An appreciable increase in anisotropy was observed upon use of non-nucleotide linker in the fluorescence probe as compared to that of the F-Probe without non-nucleotide linker.     

Detection of Neuropeptide mRNA in Rat Brain by In Situ Hybridization with Radiolabeled Synthetic Oligonucleotide Probes

Abstract

Radiolabeled synthetic oligonucleotide probes were used for detection of somatostatin and vasopressin mRNA in rat brain by in situ hybridization.     

应用寡核苷酸微阵列检测肺癌样品中的P53和K-ras基因点突变

对影响寡核苷酸微阵列检测点突变的敏感性和特异性的各种因素,如杂交液、杂交温度、标记引物浓度及其比例等,进行了研究。采用不对称PCR扩增有利于敏感性提高;多重不对称PCR不影响杂交的特异性,且敏感性有所增加。对30例肺癌标本进行寡核苷酸微阵列检测,发现12例标本发生了P53基因点突变,K-ras突变有5例。与测序结果相比,P53基因突变符合率达到80%。由于检测样本较少且检测位点不完全,因而未得到K-ras和P53基因突变与肿瘤的种类、病期及吸烟之间的明显相关性。     

应用寡核苷酸微阵列检测肺癌样品中的P53和K-ras基因点突变

对影响寡核苷酸微阵列检测点突变的敏感性和特异性的各种因素,如杂交液,杂交温度,标记引物浓度及其比例等,进行了研究,采用不对称PCR扩增有利于敏感性提高,多重不对称PCR不影响杂交的特异性,且敏感性有所增加,对30例肺癌标本进行寡核苷酸微阵列检测,发现12例标本发生了P53基因来点突变,K-ras突变有5例,与测序结果相比,P53基因突变符合率达到80％,由于检测样本较少且检测位点不完全,因而未得到K－ras和P53基因突变与肿瘤的种类,病期及吸烟之间的明显相关性。     

荧光标记寡核苷酸探针及其应用

寡核苷酸探针的标记非常重要。近年来 ,用荧光染料对探针进行非放射性标记受到很大重视 ,并取得了迅速发展 ,广泛应用于核酸序列测定、基因检测以及疾病诊断等。以下就寡核苷酸探针的荧光标记及其应用作一简要综述。     

Specific Detection of Dehalococcoides Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes

The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.     

High-Resolution and Specific Detection of Bacteria on Complex Surfaces Using Nanoparticle Probes and Electron Microscopy

The study of the interaction of bacteria with surfaces requires the detection of specific bacterial groups with high spatial resolution. Here, we describe a method to rapidly and efficiently add nanogold particles to oligonucleotide probes, which target bacterial ribosomal RNA. These nanogold-labeled probes are then used in an in situ hybridization procedure that ensures both cellular integrity and high specificity. Electron microscopy subsequently enables the visualization of specific cells with high local precision on complex surface structures. This method will contribute to an increased understanding of how bacteria interact with surface structures on a sub-micron scale.     

人乳头瘤病毒芯片寡核苷酸探针的研究

高危型人乳头瘤病毒（Human papillomavims,HPV）是宫颈癌的主要致病因子。利用Arraydesigner2．0和BLAST等生物学软件对10种型别的人乳头瘤病毒全基因组序列进行分析,设计高特异性、熔解温度（Tm）和GC含量相近的60mer HPV型特异性寡核苷酸探针,用于HPV检测芯片的制备,并对其中四型最常见HPV病毒（HPV6,11,16,18）探针的有效性进行初步验证,结果表明设计所得的探针型特异性好,可以应用于HPV的检测与分型。     

Oligonucleotide Probes for the Detection of Representatives of the Genus Thermoanaerobacter

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection of representatives of the genus Thermoanaerobacter. To increase the specificity of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5"-GCTTCCGCDYCCCACACCTA-3") detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5"-TTAACTACGGCACGRAATGCTTC-3") was specific for the first group of species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5"-CACTAMYGGGGTTTACAACC-3") targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5"-TCCTCCATCAGGATGCCCTA-3") was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).     

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.     

Detection of Hyperthermophilic Archaea of the Genus Desulfurococcus by Hybridization with Oligonucleotide Probes

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a designed Crenarchaeota-specific primer pair: Cren 7F (5"-TTCCGGTTGATCCYGCCGGACC-3") and Cren 518R (5"-GCTGGTWTTACCGCGGCGGCTGA-3"). The PCR products were hybridized with Dig-11-dUTP–labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5"-CGTTAACYCCYGCCACACC-3") and its species D. mobilis (Dco_mob 198, 5"-CGTTAACCCCTGCCACACC-3") and D. amylolyticus (Dco_amy 198, 5"-CGTTAACCCCCGCCACACC-3"). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the speciesDesulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

时间分辨荧光分析法在DNA探针检测中的初步应用

应用时间分辨荧光技术进行核酸杂交分析,选用自制整合剂异硫氰酸苯基-EDTA将铕离子标记连接于链霉亲和素分子中,通过光化学反应制备生物素标记pUC118DNA探针,与固定在聚苯乙烯微滴板中的靶DNA杂交后,以铕离子Eu(3+)标记的链霉亲和素为检测物,检测靶DNA的含量,可检测到30pg的靶DNA．     

宫颈癌组织人乳头瘤病毒的荧光偏振基因分型

采用荧光偏振人乳头瘤病毒(human papillomavirus,HPV)分型新方法探讨了8种常见型别HPV在陕西宫颈癌患者中的流行情况。首先,用HPV GP5 ／GP6 通用引物PCR扩增65例早期宫颈癌(Ⅱa期内)和72例慢性宫颈炎病变组织DNA粗提物,继之将模板指导的末端延伸反应与荧光偏振检测技术结合(TDI-FP),用GP5 ／GP6 扩增区内的HPV6、11、16、18、31、33、35和58型特异性探针与PCR产物杂交后,荧光素标记的特异碱基(TAMRA-ddTTP或R110-ddGTP)在GP5 ／GP6 产物中相应的模板指导下,掺入延伸至相应探针末端,致使对应的TAMRA或R110 FP值升高,从而对扩增的HPV阳性产物进行HPV分型。65例宫颈癌患者中检出HPV57例,阳性率87．69％,72例慢性宫颈炎患者中检出HPV28例,阳性率38．89％,两组间HPV阳性率有显著性差异。宫颈癌与慢性宫颈炎患者中4种最常见的HPV型别分别是HPV 16(45．6％)、HPV 18(22．8％)、HPV 58(17．5％)、HPV 31(7．02％)和HPV 16(35．7％)、HPV 11(32．1％)、HPV 6(21．4％)、HPV 18(10．7％)。慢性宫颈炎患者中检出的HPV型别57．14％属高危型。HPV 16在两组中均最为多见。中国陕西宫颈疾病患者中HPV感染有其特点,世界范围内少见的HPV 58在陕西宫颈癌与慢性宫颈炎患者中均较为多见,在进行HPV新诊断方法及疫苗研制时应考虑到这种特点。     

用寡核苷酸探针（CAC）5／（GTG）5进行人的DNA指纹分析

在人或动物的基因组中,存在着类似于小卫星DNA的简单衔接重复单位,如(GACA)_4、(GATA)_4、(TCC)_5、(CA)_8和(CAC)_5等,由于这些重复单位在人或动物基因组中出现的数目和频率不同而表现出多态性。本文用人工合成的寡核苷酸探针(CAC)_5/(GTG)_5,调查了北京地区的50名无关个体,经过统计学处理,计算出无关个体的相关机率是3.8×10~(-10);此外,还对2个家系中的11名成员和1对双胞胎进行了检测,其结果显示子代中的杂交带分别来自父亲和母亲;双胞胎的DNA指纹图完全一致。研究结果表明,(CAC)_5/(GTG)_5探针检出的谱带具有高度的个体特异性(同卵双生子除外),并且谱带在亲代与子代间的传递符合孟德尔遗传规律。     

用寡聚核苷酸片段筛选白色念珠菌MAPK的基因家族

促分裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ,ＭＡＰＫ）是一类与哺乳动物ｐ３４＾ＣＤＣ２同源性很高的Ｓｅｒ／Ｔｈｒ蛋白激酶,在多个不同的信号转导途径中起作用。现有的实验语气表明,ＭＡＰＫ很可能在白色念珠菌形态发生中起作用。我们根据白色念念菌的已知的两个ＭＡＰＫ基因;ＣＥＫ１与ＭＫＣ１第Ⅶ亚结构域的核苷酸序列合成卫段２７ｎｔ的寡核苷酸,作为探针来筛选白色念穆     

化学法合成生物素标记寡核苷酸探针

 生物素可通过氨烷基磷酰胺基团连接到寡核苷酸5'末端,反应在水溶液中进行,核苷酸的侧链基团不用保护。我们以这种化学法合成了生物素标记的寡核苷酸探针,其显色灵敏度达50pg,杂交灵敏度达0.4fmol,并与酶标生物素寡核苷酸探针进行了比较,也对两种不同显色体系作了比较。