地高辛标记探针检测Vero细胞狂犬病疫苗残余Vero细胞DNA含量

地高辛标记Vero细胞DNA,并制备成DNA探针,以此探针进行点杂交,确定探针的工作浓度,特异性及灵敏度,并用此法监测Vero细胞狂犬病疫苗浓缩原液纯化工艺的Vero细胞DNA去除率,测定精制Vero细胞狂犬病疫苗成品中残余Vero细胞DNA含量,结果明显,该法特异性强,重复性好,快速简便,灵敏度高,检测值可达5pg/剂量,可用于精制ero细胞狂犬病疫苗纯化工艺的质量控制和半成品检定。     

地高辛标记探针检测生物制品中残余DNA含量

用地高辛标记探针检测由传代细胞系生产的人用精制狂犬病疫苗,重组（CHO细胞）乙肝疫苗,出血热疫苗及痢疾多糖结合疫苗原液中残余DNA含量。结果表明,该方法特异性强,灵敏度高,可用于上述生物制品中残余DNA含量的检测。     

地高辛标记探针检测重组定点突变巴曲酶蛋白宿主DNA残留量的研究

目的 建立检测重组定点突变巴曲酶原液中外源性DNA残留量的方法.方法 从重组定点突变巴曲酶酵母工程菌提取基因组DNA作为模板,制备地高辛标记探针.此探针与样品进行点样杂交,并进行显色反应.结果 3批重组定点突变巴曲酶原液中,每人份剂量的宿主DNA残留量均＜10 ng.结论 该方法检测灵敏度较好,特异性较强,可用于重组定点突变巴曲酶的检测.     

人γ型基因工程干扰素中残余DNA的检测

采用地高辛标记探针,以核酸杂交法检测人γ型基因工程干扰素(IFN-γ)制品中残余DNA含量,结果表明,自制的二组DNA标准品相应量间显示的色斑相差悬殊,提示高蛋白含量对痕量残余DNA的检测干扰较大,添加IFN-γ的DNA标准品比纯DNA标准品更合理,以此测得各样品均符合WHO要求(<100pg/剂量),方法敏感性为4pg.     

地高辛探针检测重组肝靶向干扰素宿主DNA残留量的研究

目的:建立检测重组肝靶向干扰素宿主DNA残留量的方法,根据此法检测3批重组肝靶向干扰素每人份剂量宿主DNA残留量。方法:以重组肝靶向干扰素工程菌基因组DNA为模板,并制备探针,用此标记探针对3批重组产品进行DNA残留量检测。结果:3批次重组肝靶向干扰素原液每人份剂量宿主DNA残留量均小于10 ng。结论:地高辛标记探针斑点杂交检测DNA残留量具有特异性强,灵敏度高,重现性好,操作简单,能快速高效检定重组肝靶向干扰素原液并对其制备过程进行质量监控。     

人巨细胞病毒的生物素DNA探针的制备和应用

本研究用克隆的HCMV AD169株DNA片段,制备了生物素标记的DNA探针,建立了检测临床脐带血、尿标本中HCMV DNA的核酸探针杂交方法。该探针可测出100pg同源DNA,不与人胚肺细胞、Hep-2细胞DNA以及其他疱疹病毒的DNA发生反应。用核酸杂交方法检测了30份脐带血标本,有11例阳性,阳性率为33％。10例孕妇尿标本中,3例阳性,阳性率为30％。检测结果表明：我们建立的生物素标记的HCMV DNA探针的点杂交法,具有高度的特异性、敏感性,比分离病毒法更迅速,可用于HCMV感染的临床标本的病毒核酸检测。     

光敏生物素和DIG标记Orf病毒DNA探针的研究

目的：是用于Orf病毒病的诊断和相应重组菌的检测,将HCE DNAHindⅢ/A,B片断及重组质粒pGRL-4A中消化的A片断用光敏生物素和地高辛进行标记,对不同病毒分离株DNA进行检测,并对不同重组质粒进行了Dot-blotting和Southem-blotting检测。结果：用光敏生物素标记的探针最低可检出12pg的DNA,地高辛标记的探针最低可检出0.1pg的DNA,且有极强的特异性。     

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。     

非洲绿猴肾细胞株AGMr（HindⅢ）—1高度重复顺序的克隆及初步应用

从非洲绿猴肾细胞株分离高度重复顺序AGMr（HindⅢ）－1基因,以pUC18质粒为载体构建重组质粒pUC18／AGMr（HindⅢ）－1,转化E．coliJM83。酶切分析、SouthernBlot分析和序列分析均证明克隆成功,但所用的第165代Vero细胞株与另一非洲绿猴肾BSC－1细胞株的序列相比较,172个碱基中有6个位点突变。用该重组质粒作探针,对Vero细胞培养的狂犬疫苗作SouthernBlot分析表明,未经纯化的半成品中残余细胞DNA长度约400～5000bp。斑点杂交分析表明,杂交特异性良好,残余细胞DNA最小检出量约4pg。提示AGMr（HindⅢ）－1高度重复顺序可特异地应用于Vero细胞残余DNA检测。     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

Application of DNA methyltransferases in targeted DNA methylation

Taxol is a valuable plant-derived drug showing activity against various cancer types. Worldwide efforts had been made to overcome the supply problem, because the supply by isolation from the bark of the slow-growing yew trees is limited. Plant cell cultures as well as chemical and biotechnological semisynthesis are processes, which are intensively investigated for the production of taxanes paclitaxel (Taxol) and docetaxel (Taxotere) in the last few years. This article provides a comparison of the current research on taxane biosynthesis and production in yew cell cultures.     

Accuracy of DNA polymerase-alpha in copying natural DNA

The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.     

Function of DNA Polymerase III in DNA Replication

RECENTLY an in vitro system for DNA replication has been described. This system could be divided into two fractions (A and B) both of which are necessary for proper DNA replication¹. Fraction A, the “soluble” fraction, contains those proteins which do not tightly bind to membranes or native DNA. Fraction B, the “insoluble” fraction, consists of DNA and membranous structures and proteins which are bound to either of them. It was shown that the soluble fraction contains at least one component which is needed at about in vivo concentration¹. Studies of one such component are described in the following.     

DNA ligases in the repair and replication of DNA

DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA.Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+). Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.     

Present-Day DNA Contamination in Ancient DNA Datasets

Present-day contamination can lead to false conclusions in ancient DNA studies. A number of methods are available to estimate contamination, which use a variety of signals and are appropriate for different types of data. Here an overview of currently available methods highlighting their strengths and weaknesses is provided, and a classification based on the signals used to estimate contamination is proposed. This overview aims at enabling researchers to choose the most appropriate methods for their dataset. Based on this classification, potential avenues for the further development of methods are discussed.