Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

二核苷酸重复多态性的非同位素检测及其在基因诊断中的应用

本文报道了一种检测二核苷酸重复多态性的简便的非同位素法,利用重复序列两侧的特异引物进行ＰＣＲ扩增,产生的等位片段在薄层变性聚丙烯酰胺凝胶电泳上分离,再用灵敏的银染法显色。该方法不需要标记ＰＣＲ产物,简便、快速,分辨率可达１ｂｐ,并可用多对引物同时进行多重ＰＣＲ分析。用此方法对ＤＭＤ家系成员ｄｙｓｔｒｏｐｈｉｎ基因的５＇－脑型外显子止游区和３＇－非翻译区的两个（ＣＡ）。位点进行了扩增片段长度多态性分     

抗原捕获聚合酶链式反应（AC－PCR）直接分型检测单纯疱疹病毒

用抗单纯疱疹病毒（ＨＳＶ）型共同性ｇＣ和ｇＤ羊克隆抗体（ＭｃＡｂ）,包被即Ｅｐｐｅｎｄｏｒｆ管,捕捉ＨＳＶ,同时加入３个引物：一个是ＨＳＶ─１／ＨＳＶ─２型共同性上游引物,另两个分别是ＨＳＶ─１和ＨＳＶ─２型特异性下游引物。借此建立了能直接分型检测ＨＳＶ的抗原捕获聚合酶链式反应（ＡＣ─ＰＣＲ）。ＨＳＶ─１的扩增产物为４７７ｂｐ,ＨＳＶ─２的为３９９ｂｐ两型病毒经ＡＣ─ＰＣＲ扩增后产生分子量不同的ＤＮＡ片段,致使ＡＣ─ＰＣＲ能直接分型检测ＨＳＶ。ＨＳＶ─１和ＨＳＶ─２扩增产物的克隆和序列分析表明,本方法特异性好。用本法检测Ｂａｌｂ／ｃ幼鼠中枢神经系统ＨＳＶ感染的脑标本,进一步证实本方法不仅敏感、特异,而且分型准确。     

应用PCR技术检测细小病毒H－1DNA在人肝癌与裸鼠正常组织中复制的差异

应用ＰＣＲ技术检测细小病毒Ｈ－１ＤＮＡ在人肝癌与裸鼠正常组织中复制的差异黄青山,马承武,郭兰萍,陈献华,罗祖玉（上海复旦大学生理与生物物理学系,上海２００４３３）关键词：自主性细小病毒Ｈ－１及ＭＶＭ,聚合酶链式反应（ＰＣＲ）,人肝癌模型,抑瘤作用,肿...     

表达HSV－2 gD基因的重组痘苗病毒保护小鼠对抗致死量HSV病霉攻击

利用ＰＣＲ方法对单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）基因进行了修饰,在其５＇端删去约５００ｂｐ的非编码区,仅保留ＡＴＧ上游７个ｂｐ。将修饰后的ＨＳＶ－２ｇＤ基因插入到带有痘苗病毒天坛株ＴＫ基因区段的痘苗表达质粒ｐＪＳＡ１１７５,置于痘苗病毒Ｐ７．５ｋ早／晚期启动子控制下。将此重组质粒用脂质体Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ￣＋痘苗病毒天坛株感染的ＴＫ￣－１４３细胞,通过同源重组机制和标志基因ＬａｃＺ产物的蓝斑显色作用,以及ＢｕｄＲ试剂对ＴＫ表型的选择压力,筛选出整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。Ｓｏｕｔｈｅｍ杂交表明,ＨＳＶ－２ｇＤ基因已正确地插入痘苗病毒ＴＫ基因区内;间接免疫荧光检测显示,ＨＳＶ－２ｇＤ蛋白已得到有效表达,且主要分布于细胞膜。重组病毒免疫家兔可产生明显的抗ＨＳＶ－２ｇＤ中和抗体。用重组病毒免疫小鼠,３周后可使９４％（１７／１８）的小鼠对抗ＨＳＶ－２的致死量攻击,表明重组病毒具有明显的免疫保护作用。     

HBV─C基因探针的制备及应用

应用ＰＣＲ技术扩增出ＨＢＶＤＮＡＣ基因片段并与ｐＡＴ１５３质粒重组,转化到Ｅ．ｃｏｌｉＲＲＩ中,经体内扩增,提纯,用光生物素标记,制备了Ｃ基因的重组质粒探针。该探针检测灵敏度在Ｓｏｕｔｈｅｒｎ印迹中达１ｐｇ,在点印迹中为５ｐｇ。用此探针以Ｓｏｕｔｈｅｒｎ印迹方式配合ＰＣＲ技术检测乙肝病人血清中的ＨＢＶＤＮＡ,在５３例ＰＣＲ产物电泳检测阴性的样品中,Ｓｏｕｔｈｅｒｎ杂交又检出１８例阳性。     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

Encephalitozoon cuniculi Isolated from the Urine of an AIDS Patient, which Differs from Canine and Murine Isolates

ABSTRACT. A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi . However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi , with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.     

Increased reliability of selective PCR by using additionally mutated primers and a commercialTaq DNA polymerase enhancer

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3′ end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selectivePCR.     

Comparative studies on the dynamics of crosslinked insulin

Molecular dynamics simulations were carried out on an insulin crosslinked between the N-terminal A chain and the C-terminal B chain to form a so-called mini-proinsulin: N -^A1-N -^B29-diaminosuberoyl insulin (DASI). To investigate the influence of crosslinking on the dynamics of the insulin moiety, the bridge was removed from a transient DASI structure and simulation was carried on independently with the then unlinked (ULKI) as well as with the crosslinked species. The effects of crystal packing and quaternary interactions were checked by simulating both types of monomers and dimers known from the hexamer structure. All simulations were compared to previous ones of native insulin. DASI shows general similarity to the native simulations in most parts of the structure. Deviations are visible in the segments to which the bridge is directly connected, i.e. their flexibility is reduced. Upon removal of the bridge the ULKI simulations reapproach those of native insulin. The influence of the bridge spreads over the whole molecule, but all of its main structural features remain intact. The simulations suggest that the displacement of the C-terminal B chain of native insulin, considered important for receptor interaction, is prevented by the bridge, which also partially shields some binding residues. This is in accordance with the poor biological potency of A1-B29-crosslinked insulins.Abbreviations DASI-insulin(DASI) bovineN -^A1-N -^B29-di-aminosuberoyl insulin - ULK-insulin (ULKI) Native beef insulin with the bridge of DASI removed