mRNA差异显示技术中特异条带回收方法的比较

目的：建立简化的mRNA差异显示技术中特异条带的回收方法。方法：用单个小鼠早期发育的胚胎构建mRNA差异显示技术,用一步法和煮沸法分别从银染的聚丙烯酰胺凝胶中回收差异显示的特异条带,并进行再扩增、回收、克隆及酶切鉴定。结果：两种不同的回收方法经过mRNA差异显示技术程序环节,证明其具有相同的实验效果。结论：应用简化的一步法和煮沸法对单胚构建的mRNA差异显示技术中的特异条带进行回收,具有有效性和可靠性。     

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer.     

Genome physical mapping with large-insert bacterial clones by fingerprint analysis: methodologies, source clone genome coverage, and contig map quality

Genome physical mapping with large-insert clones by fingerprint analysis is becoming an active area of genomics research. Here, we report two new capillary electrophoresis-based fingerprinting methods for genome physical mapping and the effects of different fingerprinting methods and source clone genome coverage on quality physical map construction revealed by computer simulations and laboratory experiments. It was shown that the manual sequencing gel-based two-enzyme fingerprinting method consistently generated larger and more accurate contigs, followed by the new capillary electrophoresis-based three-enzyme method, the new capillary electrophoresis-based five-enzyme (SNaPshot) method, the agarose gel-based one-enzyme method, and the automatic sequencing gel-based four-enzyme method, in descending order, when 1% or fewer questionable clones were allowed. Analysis of clones equivalent to 5x, 8x, 10x, and 15x genomes using the fingerprinting methods revealed that as the number of clones increased from 5x to 10x, the contig length rapidly increased for all methods. However, when the number of clones was increased from 10x to 15x coverage, the contig length at best increased at a lower rate or even decreased. The results will provide useful knowledge and strategies for effective construction of quality genome physical maps for advanced genomics research.     

cDNA-AFLP display for the isolation of Peronospora parasitica genes expressed during infection in Arabidopsis thaliana

To identify genes from the obligatory biotrophic oomycete Peronospora parasitica that are expressed during infection in Arabidopsis thaliana we employed cDNA-amplified fragment length polymorphism (AFLP) display. cDNA-AFLP fragments from infected and non-infected leaves were separated in parallel by gel electrophoresis and displayed by autoradiography. Most differential gene fragments were derived from P. parasitica.     

Bacterial artificial chromosome-based physical map of the rice genome constructed by restriction fingerprint analysis

Genome-wide physical mapping with bacteria-based large-insert clones (e.g., BACs, PACs, and PBCs) promises to revolutionize genomics of large, complex genomes. To accelerate rice and other grass species genome research, we developed a genome-wide BAC-based map of the rice genome. The map consists of 298 BAC contigs and covers 419 Mb of the 430-Mb rice genome. Subsequent analysis indicated that the contigs constituting the map are accurate and reliable. Particularly important to proficiency were (1) a high-resolution, high-throughput DNA sequencing gel-based electrophoretic method for BAC fingerprinting, (2) the use of several complementary large-insert BAC libraries, and (3) computer-aided contig assembly. It has been demonstrated that the fingerprinting method is not significantly influenced by repeated sequences, genome size, and genome complexity. Use of several complementary libraries developed with different restriction enzymes minimized the "gaps" in the physical map. In contrast to previous estimates, a clonal coverage of 6.0-8.0 genome equivalents seems to be sufficient for development of a genome-wide physical map of approximately 95% genome coverage. This study indicates that genome-wide BAC-based physical maps can be developed quickly and economically for a variety of plant and animal species by restriction fingerprint analysis via DNA sequencing gel-based electrophoresis.     

应用改良的mRNA差异展示法克隆人鼻咽上皮特异性表达的cDNA片段

本文对ｍＲＮＡ差异展示法进行了改良．利用一组随机引物与来源于ｍＲＮＡ翻译起始位点区域的引物组合进行ＲＴ－ＰＣＲ,使差异展示的片段来源于蛋白编码区,并对差异展示的条件进行了优化．应用此法对人鼻咽上皮与软腭口腔粘膜上皮的基因表达进行了比较研究,得到了１０个在鼻咽上皮特异表达的ｃＤＮＡ片段,并对其中的５个片段进行了亚克隆和序列分析．通过与ＧｅｎｅＢａｎｋ数据库中的序列进行同源性比较,确定其中两个片段为未知新序列,Ｎｏｒｔｈｅｒｎ杂交证实其中一个片段ＮＥＳ１为鼻咽上皮特异性表达片段．     

cDNA-AFLP和cDNA-SRAP技术在植物基因差异表达上的应用及其分析比较

对cDNA-AFLP和cDNA-SRAP技术在植物上的应用研究进行了介绍,指出cDNA-AFLP和cDNA-SRAP技术已逐渐成为基因表达研究的重要工具,是构建转录图谱、定位目标性状位点、研究差异基因表达等研究的主要手段,具有广泛广阔的应用前景.但是cDNA-AFLP和cDNA-SRAP技术均具有一定的缺限,主要表现在获得的差异片段还不够全面丰富.cDNA-AFLP可以扩增到扩增较多的带,但试验程序比较繁琐、昂贵而且耗时较长、操作较困难;而cDNA-SRAP相对比较简便、便宜而耗时较短,较易操作.因而将cDNA-AFLP与cDNA-SRAP技术结合起来,可望得到更全面丰富的差异片段,从而在植物基因差异表达、新基因发现、抗逆性分子机理研究等方面发挥更大的作用.     

克隆差异表达基因的新策略

基因表达的变化有两种,即新出现的基因表达与表达量差异的基因表达.表达量差异的基因克隆技术主要有mRNA差异展示,此技术是目前筛选差异表达基因最有效的方法之一,但主要存在假阳性率高的不足,针对此缺点,近几年提出了新的策略与方法,如差异消减展示、基于PCR和减法杂交基础上的差异表达基因克隆技术,这些技术具有显著优势.     

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining

Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.     

New protein extraction/solubilization protocol for gel-based proteomics of rat (female) whole brain and brain regions

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using pre-cast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, in-gel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.     

Isolation of poly (A)+ mRNA for downstream reactions from some medicinal and aromatic plants

In the present protocol for extraction of RNA, hexadecyltrimethylammoniumbromide (CTAB) and insoluble polyvinylpyrrolidone were used followed by LiCl precipitation, CsCl ultracentrifugation and finally poly (A)+ mRNA was isolated with the help of oligo(dT)-cellulose columns. The isolated poly (A)+ mRNA was found to be suitable for cDNA-AFLP and suppression subtractive hybridization applications. It is a modified and consolidated protocol based on previously described methods for isolated steps and works better for medicinal and aromatic plants. High yield of poly (A)+ mRNA coupled with its amenability for downstream reactions like RT-PCR, northern blotting and cDNA synthesis for library construction is a key feature of the present protocol.     

植物铝胁迫响应基因的研究进展

铝毒是酸性土壤中植物生长和作物生产的主要限制因子.近年来的很多研究应用差异显示PCR、抑制差减cDNA文库和DNA微正列等技术,在一些铝耐受型和敏感型植物中鉴定了很多铝胁迫响应基因.本研究通过参阅国内外有关报道和结合本实验室的研究成果,从铝诱导的通道蛋白、代谢相关、胁迫和细胞死亡以及信号转导相关基因4个方面的研究进展进行了综述.