用PCR法直接快速筛查重组阳性克隆

应用ＰＣＲ法快速筛查插入有苯丙氨酸脱氨酶ｃＤＮＡ重组阳性克隆。方法：用于ＰＣＲ扩增的引物是位于载体ｐＥＴ２３ｂ启动子处的Ｔ７启动子引物和位于目的基因ＰＡＬｃＤＮＡ３’端终止密码ＴＡＡ处的引物。以灭菌吸头挑一单菌落加入ＰＣＲ体系扩增。结果：在筛查的３个克隆中,有２个阳性克降,并且插入方向正确,经ＤＮＡ序列测定得到进一步证实。结论：以ＰＣＲ方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方面,不     

牛β-酪蛋白5'端上游调控序列的克隆和序列分析

该文用ＰＣＲ扩增了牛β－酪蛋白基因５‘－端上游调控序列,并对其进行了克隆和序列分析。采集成年母牛肝,提取ＤＮＡ。在牛β－酪蛋白基因外显子１和上游调控区内设计引物,扩增其上游调控序列。两条引物长均为１９个核苷酶,引物间跨度为６３５ｂｐ。以牛肝ＤＮＡ为模板,进行ＰＣＲ扩增,扩增产物在２％琼脂糖凝胶上电泳,可见特异的目的条带。     

国内信息

ＤＥＧＣＭ技术中的ＲＤＡ、ＳＳＨ和ＲＳＤＤ差异表达基因分离技术 (ＤＥＧＣＭ )发展很快 ,主要是ＰＣＲ技术的发展和相应新技术的出现为ＤＥＧＣＭ注入了新的活力。ＰＣＲ引入差式筛选之中 ,可提高效力和降低假阳性率。若引入扣除杂交 ,可发展扣除扩增方案 ,并用特异引物结合于ＰＣＲ中选择扩增差异ｃＤＮＡ而形成代表性差式分析法 (ＲＤＡ)。采用抑制性ＰＣＲ选择性扩增差异基因片段 ,用ＤＮＡ链内“退火”优先于链间退火 ,使非目的序列产生“发夹”式互补结构 ,不能与引物配对 ,从而选择性抑制非目的基因片段的扩增 ,以此形成抑制性差…     

邻单胞菌邻苯二酚1,2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达

根据已报道的邻苯二酚１,２－双加氧酶基因（ｔｆｄ Ｃ）序列,设计ＰＣＲ引物,从一株邻单胞菌（Ｐｌｅｓｉｏｍｉｎａｓ）的ｐＬ１质粒上扩增到ｔｆｄ Ｃ基因片段,连接到ｐＧＥＭ－Ｔ载体上,并转化大肠杆菌ｌＭ１０９菌株,筛选到阳性克隆。序列分析结果表明,ＰＣＲ产物全长８０１ｂｐ,有一阅读框,编码２５５个氨基酸,与增氧产碱菌（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｌｕｓ）的ｔｆｄ Ｃ基因相比,在６９３位相差一     

原位杂交及原位PCR检测幽门螺杆菌

本研究建立了检测幽门螺杆菌的原位杂交和原位ＰＣＲ技术,采用ＰＣＲ掺入的方法标记原位杂交的生物素探针,６份ＨＰ阳性胃活检组织冰冻切片杂交均为阳性,６份阴性对照组织为阴性。９／１２份ＨＰ阳性对照组织石蜡切片杂交阳性,２份阴性对照切片为阴性。３份阳性对照猫胃粘膜冰冻切片杂交也是阳性。原位ＰＣＲ的引物来自ＨＰ的１６ｓｒＲＮＡ基因,在扩增过程掺入生物素基因。４／４例ＨＰ阳性人胃粘膜冰冻切片原位扩增阳性,２／     

用DD—PCR法克隆AVP4—8诱导的鼠脑差异表达基因

精氨酸加压素（ＡＶＰ）Ｃ端片段ＡＶＰ４－８,具有增强记忆的功能,它在大鼠脑内引发一系列的生理和生化变化。采用差示ＰＣＲ（ＤＤ－ＰＣＲ）技术,寻找注射ＡＶＰ４－８前后在大鼠海马中差异表达的基因。抽提总ＲＮＡ,以反转录产生的ｃＤＮＡ为模板进行ＰＣＲ,经过９组引物组合,获得了十几个差异片段,挑选其中差异最大的片段ｄｄｌ进行克隆,测序,并与Ｇｅｎｅｄａｎｋ等数据进行同源比较 有找到同源基因,可能是个新基因     

中国人肥胖基因真核表达载体的构建

为研究肥胖（ｏｂｅｓｉｔｙ）的病因及肥胖基因（ｏｂ）的表达与调控,根据文献报道的ｈｏｂ序列设计引物,经ＲＴ－ＰＣＲ扩增中国人的ｏｂ基因（包括信号肽在内的ｃＤＮＡ全长５４０ｂｐ）,ＰＣＲ产物采用Ｔ－Ａ克隆法首先连接到克隆载体ｐＵＣ１１９,然后定向转移到经改造的真核表达载体ｐＳＶ－β－ｌａｃＺ,酶谱分析表达克隆基因为的人肥胖基因。     

RPC技术检测儿童龈炎中的卟啉单胞菌

根据牙龈卟啉单胞菌特异的纤毛亚单位蛋白结构基因,设计一对寡核苷酸引物,采用ＰＣＲ扩增了１３１ｂｐ特异片段,实验证明,ＰＣＲ直接检测临床标本中的牙龈卟啉单胞菌,不仅特异、敏感、而且快速,从而显示了较好的优越性。用该引物,分析牙龈卟啉单胞菌在儿童龈炎中的分布。经ＰＣＲ检测４６例龈下菌斑标本中的牙龈卟啉单胞菌,结果表明：２４例标本ＰＣＲ为阳性;对照组４６例标本中,牙龈卟啉单胞菌仅有５例为阳性,儿童龈炎中牙龈卟啉单胞菌的检出率明显高出正常组（ｐ＞０００５）。提示用ＰＣＲ检测儿童龈炎中的牙龈卟啉单胞菌具有重要意义。     

橡胶延伸因子基因及3′端非编码区的克隆与序列分析

橡胶延伸因子ＲＥＦ（Ｒｕｂｂｅｒ Ｅｌｏｎｇａｔｉｏｎ Ｆａｃｔｏｒ）是橡胶生物合成中最重要的酶之一,作者利用ＲＥＦ基因序列设计并合成引物。通过提取胶乳总ＲＮＡ用ＲＴ法合成ｃＤＮＡ后,通过ＰＣＲ技术坟民并克隆了ＲＥＦ基因和３′端非编码区,序列测定结果表明,ＲＥＦ基因全长４１４个碱基,编码１３８个氨基酸,３′端非编码区长１１２ｂｐ．与ＧｏｙｖａｅｒｔｓＥ等人发表的序列比较：ＲＥＦ基因同源率为１００％     

粘虫核型多角体病毒919bp片段的PCR双链直接序列测定

本文发展了ＰＣＲ克隆和亚克隆技术制备ＤＮＡ测序模板。首先,我们用ｐＵＣ／Ｍ１３系列质粒的通用正反向引物ＰＣＲ扩增出质粒ｐＢｌｕｅｓｃｒｉｐｔＫＳＤＮＡ的多克隆位点及其侧翼序列,用ＥｃｏＲＶ和ＸｈｏＩ消化成为左右两个引物多克隆臂,与粘虫核型多角体病毒（ＬｓＮＰＶ）的ＥｃｏＲＶ和ＸｈｏＩ约４００ｂｐ和５００ｂｐ片段分别连接,经ＰＣＲ扩增,得到两端具有上述正反向引物结合位点的测序模板,用ｄｄＮＴＰ链终止法／ＰＣＲ扩增／银染色,从片段两端测定了全部９１９ｂｐ序列,这种ｄｄＮＴＰ／ＰＣＲ／银染测序法简化了操作,大大缩短了测序模板的制备时间,易于实现自动化操作。     

PCR in situ: aspects which reduce amplification and generate false-positive results

Summary PCRin situ promises the ability to amplify and detect very low levels of target nucleic acid in tissues. Despite considerable effort, the technique is still technically difficult and has not yet proved to be reliable or reproducible. We have now identified a number of factors which can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the effects of fixation; reagent abstraction, DNA degradation, DNA end-labelling and product diffusion. We present evidence to show that formaldehyde fixation cross-links histones to DNA and thus restricts the subsequent amplification of target sequences by PCR. End-labelling of DNA occurs when direct incorporation is used to detect amplified products and this gives rise to false-positive signals. Amplified products can also diffuse out of cells and into neighbouring cells which do not contain target sequences. They can undergo re-amplification within these cells giving rise to false-positive signals. We believe considerable caution should be exercised in the interpretation of results generated using PCRin situ.     

Screening of recombinant Escherichia coli using activation of green fluorescent protein as an indicator

A novel cloning vector that can be used to identify recombinant Escherichia coli colonies by activation of the green fluorescent protein gene (GFP) was constructed. Screening using the vector does not require special reagents. The recombinant plasmid activates GFP, and the rate of false-positive results is low.     

GFP为报告基因的酵母哺乳动物细胞穿梭载体的构建及其在人血管内皮细胞中的表达

为研究酵母作为载体在口服基因治疗及免疫中的作用 ,需要一种能够在酵母中复制而在哺乳动物细胞中表达的穿梭载体 .利用通用载体质粒融合系统 (UPS)构建了一种以GFP为报告基因的新载体 ,以常规的氯化锂法对酿酒酵母进行转化 ,证明该载体能够在酵母中复制 ;以脂质体介导向人血管内皮细胞进行了转染 ,有绿色荧光 ,证明该载体能够在哺乳动物细胞中表达 .所获得的新型的穿梭载体为口服酵母在基因治疗中的应用提供了物质准备     

Polymerase chain reaction in situ: an appraisal of an emerging technique

Summary Polymerase chain reaction (PCR)in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCRin situ is, however, technically difficult, and amplification of the target DNA is only 30–300 fold. In this article we present an overview of PCRin situ techniques used to amplify both DNA and RNA targets (RT-PCRin situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks byTaq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.     

Development of a Rapid Method for Identifying Carryover Contamination of Positive Control DNA,Using a Chimeric Positive Control and Restriction Enzyme for the Diagnosis of White Spot Syndrome Virus by Nested PCR

Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447 bp; WSSV 2nd PCR product, 941 bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1 h at 37 °C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.     

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.     

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.     

Clearance of false-positive antigen-antibody reactions of a diagnostic antigen produced inEscherichia coli with human sera

Although many pharmaceutically useful proteins are produced inE. coli expression system, it is very rare for the system to be used in the production of diagnostic antigen due to a major problem,i.e., false-positive reaction ofE. coli host-derived proteins contaminating purified diagnostic antigen with human sera. The N (nucleocapsid) protein of Seoul virus causing haemorrhagic fever with renal syndrome (HFRS) was produced inE. coli BL21 (DE3), and used for the detection of N protein-specific antibodies in human sera. Using the N protein as a diagnostic antigen of HFRS, the false-positive reaction was cleared by merely mixing the test sera with the extract ofE. coli host strain not harboring expression plasmid.     

携带IL-2/NK4双基因减毒沙门氏菌TPIN的遗传稳定性研究

对携带IL-2/NK4双基因减毒沙门氏菌TPIN的遗传稳定性进行研究。将TPIN连续传40代,取10代、20代、30代、40代菌落进行鉴定,包括菌落和菌体的形态、质粒的纯度和浓度、目的基因片段、双酶切结果及转染人肝癌细胞(HepG2)后目的基因体外表达活性。结果表明,每10代的菌落和菌体形态一致,其所含质粒的纯度和浓度在传代过程中无明显变化,PCR扩增的目的基因条带大小一致,双酶切鉴定结果正确,转染HepG2细胞后,每代间所表达目的蛋白浓度无明显差异。TPIN作为消化道肿瘤的生物治疗药物,在遗传方面有较好的稳定性,可进一步进行大量生产。     

Plasmid structural instability associated with pC194 replication functions

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When gram-positive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S.D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested. It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.