反选标记法在微生物基因敲除中的应用

随着后基因组时代的到来,简单高效的基因编辑工具和方法在研究微生物的基因表达调控、工程菌株的遗传改造等方面发挥了巨大作用。反选标记法是基于同源重组原理进行基因敲除的一种便捷高效的方法,该方法通过选择性的遗传标记的丢失和回补,实现对靶基因的敲除或目的基因的插入等分子遗传操作。这种选择性的遗传标记一般不依赖于传统的抗生素筛选标签,且同一反选标记能够反复使用,可连续进行多轮遗传操作,实现对多个基因靶位点的分子编辑。对近年来反选标记基因的种类、作用原理及在微生物基因敲除实验中的应用进行综述,以期为高效、可靠的分子编辑方法的研究提供参考。     

常规组织切片凋亡细胞原位末端标记方法

凋亡是有别于组织坏死的一种细胞死亡方式,多与基因调控的编程性死亡有关。准确地判断细胞凋亡对探讨程序化细胞死亡诱发机制具有重要意义。本文采用Biotin-16-dUTP对凋亡细胞内DNA断裂片段的末端进行标记,可原位检测常规组织切片上的凋亡细胞,方法具有敏感、直观、重复性好等优点。     

国内信息——五种基因克隆技术

经华中农业大学周国岭先生对国内外64篇有关基因克隆技术的论文概括和分析,当前基因克隆技术主要有5个类别,即体位克隆或状态克隆、转位或转DNA标记法、同系位候选基因法、快捷顺序标记法、差接式快捷基因克隆法等。     

玉米抗大斑病基因Ht2、Ht3分子标记的应用检测

采用包括两套近等基因系在内的11份含有不同抗大斑病基因的玉米材料,对已报道的与Ht2、Ht3基因连锁的分子标记进行应用性检测。实验选用umc1202、bnlg1152、umc1149、SD-06633和bnlg1666等5对引物进行PCR扩增检测,发现被检测引物均缺乏对相应标记基因的特异性扩增;对与Ht2基因连锁的SCAR引物SD-06633的扩增片段进行了序列分析,发现其在Ht2和HtN基因背景下的扩增片段长度均为631bp,且序列相似性高达98.73%,扩增产物特征一致,无法证明该标记的特异性。检测结果表明,上述已发表的与抗大斑病基因Ht2和Ht3紧密连锁的5个分子标记缺乏特异性,不适用于玉米材料的Ht2和Ht3基因型鉴定。     

PCR标记前胶原基因探针及其检测肝星状细胞前胶原基因表达

目的：建立一种新的前胶原基因探针制备方法,并用克勤克俭 检测HSC的前胶原mRNA表达。方法：从NCBIGeneBank查询Ⅰ、Ⅲ、Ⅳ型前胶原基因的序列,根据基因序列用OLIGO软件设计其引物：RT-PCR扩增基因,并用不对称PCR方法和DIG-dUTP标记前胶原基因探针,用其原位杂交检测HSC前胶原基因表达。结果：用所设计的引物和RT-PCR扩增得到目的基因,制备了DIG标记的前胶原基因探针,并用其检到HSC的前胶左面的基因表达。结论：建立了一种新的、较简易的前胶原基因探针标记方法,并对其它基因探针的标记有借鉴意义。     

应用RAPD标记法鉴定甘蓝型油菜杂种纯度

近年 ,华中农大作物遗传改良国家重点实验室国家油菜改良武汉分中心的在职研究生段院和刘平武等 ,对用RAPD标记鉴定甘蓝型油菜杂种H990 9的纯度作了研究和报道。他们对杂交组合H990 9两亲本的指纹图谱作了分析研究 ,选出S374、SI334、SI36 5、UBC5 2 34个引物作杂种的纯度鉴定     

地高辛配基标记核酸技术及其应用

核酸探针通过地高辛配基(异羟基洋地黄毒苷配基)标记的脱氧尿嘧啶核苷三磷酸(Dig-dUTP)随机引物插入结合而被标记,然后辅以免疫酶化学检测等新技术。和放射性同位素标记探针一样,它已广泛用于核酸等物的各种探测及分析,具有灵敏、安全、简便、经济及实用等优点。     

转基因烟草检测技术研究

本研究建立了转基因烟草常用调控基因35S启动子和nos终止子,标记基因npt-II和目的基因TMV－CP的PCR检测技术,并用轻构建的pUC18质粒提纯的35S启动子和nos终止子基因经地高辛标记后制成了基因探针,建立了检测35S启动子和os终止子的分子杂交技术,杂交试验证明,PCR扩增的35S启动子和nos终止子为特异性扩增产物,此项检测技术具有快速,特异,敏感,经济以及可重复性强等优点。     

稳定同位素标记核酸法在非培养微生物代谢功能研究中的应用

基于核糖体RNA(rRNA)序列分析的系统发育信息是研究微生物代谢功能的可靠指标之一。复杂环境样本中的微生物利用了稳定同位素标记的营养物后,分离其核酸进行序列分析或检测其核酸中同位素的丰度变化,就可以不必培养分离微生物而揭示出它们的代谢功能。     

通用引物双重PCR在节肢动物物种鉴定中的应用

【目的】本研究旨在使用基于线粒体基因通用引物的双重PCR技术同时扩增单一样本中两条标记基因,从而达到简化节肢动物物种鉴定流程的目的。【方法】在一次PCR实验中同时加入可扩增线粒体COI基因和16S rDNA两个不同分子标记的引物,对3纲8目14科的14种节肢动物物种标本的基因组DNA进行扩增;扩增产物经电泳和胶回收后测序,并BLAST在线搜索相似序列,验证基于通用引物的双重PCR在不同的动物类群中用于物种鉴定的有效性。【结果】应用基于COI和16S rDNA的引物从分属于3纲8目14科的14种节肢动物基因组DNA中均可成功扩增目的基因;扩增产物测序结果进一步证实了扩增的准确性。【结论】通过本方法进行物种的分子鉴定,不仅可以保证物种鉴定的高准确率,还可以明显减少时间与DNA样本量的消耗,这对需要快速准确鉴定物种或珍稀的材料样本十分重要。     

N’-acyl-N-methylphenylenediamine as a novel proximity labeling agent for signal amplification in immunohistochemistry

We synthesized novel phenylenediamine derivatives and evaluated them as labeling agents to label proteins in close proximity to a single electron transfer catalyst. We found that N’-acyl-N-methylphenylenediamine labels tyrosine effectively in a model experiment using tris(bipyridine)ruthenium (Ru(bpy)₃²⁺) as the single electron transfer catalyst. By changing the substituents on the nitrogen atom of the phenylenediamine derivatives, the electrochemical properties of the labeling agent can be drastically changed. On the other hand, horseradish peroxidase (HRP) also catalyzes the reaction with almost the same oxidation potential as Ru(bpy)₃²⁺ (～+1.1?V). HRP proximity labeling is applicable to signal amplification in immunohistochemistry. We evaluated the phenylenediamine derivatives as labeling agents for HRP proximity labeling and signal amplification, and found that N’-acyl-N-methylphenylenediamine is a novel and efficient agent for signal amplification using HRP in immunohistochemistry.     

PCR-mediated screening and labeling of DNA from clones

A simplified and economical protocol for DNA library screening and nonradioactive labeling is described. Bacterial clones are lysed in 1% of Triton X-100 and subjected to polymerase chain reaction in the presence of digoxigenin-11-dUTP to screen and simultaneously to label the DNA inserts. Bacteriallysates are stable in storage at −20°C and can be used repeatedly for PCR-mediated labeling. In this protocol, very low concentrations of dNTP, digoxigenin-dUTP, and primers are used in combination with a reduced reaction volume. This will considerably reduce the expense of screening and labeling bacterial clones and facilitate the exchange of DNA probes among laboratories.     

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.     

疱疹病毒定量PCR的建立

A single pair of oligonucleatide primer selected within a highly conserved region of the DNA polymerase gene in herpesviruses was synthesized. The competitive template DNA purified from cytomegalovirus (CMV) DNA was used to carry out competiitve PCR amplification with herpes simplex virus type 1 (HSV1) DNA (target sequences). And anti-HSV1 effects of acyclovir (ACV) was investigated by the method.The results showed that the efficacy of PCR amplification was equal to each other(the ratio of the quantity of c…     

链霉菌工业菌种HS007的基因组标记

通过小片段基因组文库的构建获得工业生产菌HS007的若干基因组片段,并以大肠杆菌-链霉菌穿梭质粒pHJL400为载体,构建了5个插入了特异性标记序列及抗性筛选标记的重组质粒pHJL02AFOH,pHJL07AFOH,pHJL08AFOH,pHJL10AFOH和pHJL12AFOH.利用这些质粒转化工业生产菌株HS007,获得具有特异性标记序列和相应抗性的标记菌株02-72,07-44,08-02,10-81和12-58,其中02-72和12-58的生产能力不受插入片段的影响.利用重组质粒pSP02AFOH上抗性标记两端两个FRT序列的分子内重组去除抗性标记,并以大肠杆菌一链霉菌穿梭质粒pGH112替换该质粒的载体部分,得到重组质粒pGH02FH.以pGH02FH转化标记菌株02-72,获得具有特异性标记序列而没有相应抗性的菌株02-72-36.发酵结果表明,标记片段的插入不影响菌株02-72-36的生产能力.本方法建立了链霉菌工业菌种基因组标记的技术平台.     

High-throughput genetic mapping in Arabidopsis thaliana

To facilitate rapid determination of the chromosomal location of novel mutations, we have improved current approaches to gene mapping using microsatellite length polymorphisms. The high-throughput linkage analysis method described here allows a novel gene to be tested for linkage against the whole genome of a multicellular eukaryote, Arabidopsis thaliana, in a single polyacrylamide gel. The procedure is based on the simultaneous co-amplification of 21 microsatellites in a single tube, using a multiplex PCR mix containing 21 primer pairs, each including one oligonucleotide labeled with one of three fluorescent dyes that have different emission wavelengths. The amplification products, which range in number from 21 to 42, depending on the genotype of the individual being tested, are electrophoresed in a single lane on a polyacrylamide gel. The use of an automated fragment analyzer makes it possible to perform linkage analysis on a one gel-one gene basis using DNA samples from 19 F₂ individuals obtained from an outcross involving a mutant and a wild-type that is genetically polymorphic with respect to the ecotype in which the mutant was generated. Discrimination of the amplification products is facilitated not only by labeling with different fluorochromes, but also by prior testing of different sequences for the ability to prime the amplification of each microsatellite, in order to ensure that multiplex PCR yields compatible amplification products of non-overlapping size. The method is particularly useful in large-scale mutagenesis projects, as well as for routine mapping of single mutants, since it reveals the map position of a gene less than 24 h after the F₂ individuals to be analyzed have become available. The concepts employed here can easily be extended to other biological systems. Received: 24 September 1998 / Accepted: 9 December 1998     

A Targeted Real-Time PCR Assay for Studying Naphthalene Degradation in the Environment

A quantitative real-time polymerase chain reaction (PCR) assay was developed for monitoring naphthalene degradation during bioremediation processes. The phylogenetic affiliations of known naphthalene-hydroxylating dioxygenase genes were determined to target functionally related bacteria, and degenerate primers were designed on the basis of the close relationships among dioxygenase genes identified from naphthalene-degrading Proteobacteria. Evaluation of the amplification specificity demonstrated that the developed real-time PCR assay represents a rapid, precise means for the group-specific enumeration of naphthalene-degrading bacteria. According to validation with bacterial pure cultures, the assay discriminated between the targeted group of naphthalene dioxygenase sequences and genes in other naphthalene or aromatic hydrocarbon-degrading bacterial strains. Specific amplification of gene fragments sharing a high sequence similarity with the genes included in the assay design was also observed in soil samples recovered from large-scale remediation processes. The target genes could be quantified reproducibly at over five orders of magnitude down to 3 × 10² gene copies. To investigate the suitability of the assay in monitoring naphthalene biodegradation, the assay was applied in enumerating the naphthalene dioxygenase genes in a soil slurry microcosm. The results were in good agreement with contaminant mineralization and dot blot quantification of nahAc gene copies. Furthermore, the real-time PCR assay was found to be more sensitive than hybridization-based analysis.     

Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling

Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.     

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10³ colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.     

Detection of bioterror agents in air samples using real-time PCR

Aims:  To use real-time PCR for the detection of bacterial bioterror agents in a liquid air sample containing potential airborne interferences, including bacteria, without the need for DNA extraction.
Methods and Results:  Bacteria in air were isolated after passive sedimentation onto R2A agar plates and characterized by 16S rRNA sequencing. Real-time PCR was used to identify different bacterial bioterror agents in an artificial air sample consisting of a liquid air sample and a mixture of miscellaneous airborne bacteria showing different colony morphology on R2A agar plates. No time-consuming DNA extraction was performed. Specifically designed fluorescent hybridization probes were used for identification.
Conclusions:  Fourteen different bacterial genera were classified by 16S rRNA gene sequencing of selected bacterial colonies showing growth on R2A agar plates. Real-time PCR amplification of all the bacterial bioterror agents was successfully obtained in the artificial air sample containing commonly found airborne bacteria and other potential airborne PCR interferences.
Significance and Impact of the Study:  Bacterial bioterror agents can be detected within 1 h in a liquid air sample containing a variety of commonly found airborne bacteria using real-time PCR. Airborne viable bacteria at Kjeller (Norway) were classified to the genera level using 16S rRNA gene sequencing.