Frankia基因组DNA提取及其PCR带型

本文采用溶菌酶法、CTAB法、微波（MW）法及CTAB＋MW法等4种方法提取Frankia菌CpIl基因组DNA。结果表明四种方式均可满足试验要求，其中以CTAB法及微波法最为有效。在比较提取方法与PCR带型间的关系中，发现提取上的差异不影响PCR带型的变化。

长白山赤杨丛枝菌根真菌多样性的半巢式LP-PCR-SSCP检测分析

利用半巢式LP PCR SSCP技术 ,对中国吉林长白山不同海拔生境下 3种赤杨共生丛枝菌根真菌的多样性进行检测分析 .结果表明 ,该地区赤杨属东北赤杨、西伯利亚赤杨及色赤杨共生丛枝菌根真菌在科乃至种的水平上并未随宿主的变化表现出丰富的多样性 ;3个树种在自身属的水平上与共生的球囊霉科 (Glomaceae)至少 1种AMF ,即G .intraradix ,在种的水平上表现出不相关于宿主海拔高度的某种相互选择性 .

长白山四种赤杨丛枝菌根真菌侵染多样性的巢式PCR-RFLP分析

采集中国吉林长白山不同海拔的4种赤杨根须样本,利用巢式PCR-RFLP方法检测丛枝菌根真菌(AMF)对样品的侵染情况,PCR结果经限制性内切酶分析.结果表明,赤杨根内AMF存在丰富的基因多样性.AMF的侵染有从宿主混乱性向宿主专一性发展的趋势.东北赤杨AMF的宿主专一性水平最强,球囊霉属已成为东北赤杨的优势侵染类群;其余3种赤杨的AMF则出现宿主混乱现象.宿主因素比海拔因素对AMF侵染有更重要的影响.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. (C) 2003 Elsevier B.V. All rights reserved.

几种PCR方法在克隆鸭肠炎病毒未知基因中的应用

以DEV基因组DNA为模板,用简并PCR、改良Targeted gene walking PCR、改良的热不对称交错PCR和Long-PCR,获得了5350bp、11083bp和2905bp3段DEV未知基因片段,DNA序列分析发现包含9个开放阅读框,将这些序列提交GenBank分别获得的登录号为:EF554396~EF554403。结果表明,多种PCR方法联合使用可以高效的实现对鸭肠炎病毒未知基因的克隆。

利用rep-PCR研究云南尼泊尔桤木根瘤内Frankia基因多样性

利用rep-PCR指纹技术对云南5个地区的尼泊尔桤木根瘤内Frankia菌基因多样性进行研究,实验发现Frankia菌呈现丰富的基因多样性.所有71个样品被分为11种不同的基因类型.除高黎贡山外,不同地域都有某种基因型占优势,显示Frankia菌基因型与地域有紧密关系.所观察的5个地区中,高黎贡山Frankia菌基因型种类最多,多样性指数最高,基因多样性最为丰富.结合高黎贡山地理资料及其它生物多样性相关研究,推测与高黎贡山尼泊尔桤木共生的Frankia可能作为种储备库,为其它地区的寄主植物提供了祖先菌株.图8表1参13

葡萄卷叶病毒RT-PCR检测技术

为调查葡萄植株携带葡萄卷叶病毒(GLRaV)的情况,以带毒和非带毒指示植物“品丽珠”为试验材料提取总RNA,并以此总RNA为模板进行RT-PCR扩增,建立了GLRaV的RT-PCR检测体系,并对GLRaV cDNA序列进行测定,结果成功地检测到了GLRaV。经多次试验证明,该检测结果准确可靠,可以用于GLRaV的检测。

实时荧光定量PCR及其在微生物生态学中的应用

定量描述微生物群落的组成,在微生物生态学的许多研究领域都是非常重要的。然而由于可培养技术的局限性,定量描述微生物群落成为比较困难的事情。最近包括PCR技术在内的分子生物学技术为人们提供了有力的工具,使对微生物群落的分布、丰度等有了进一步的了解。实时荧光定量PCR技术作为核酸定量检测技术,自从发明以来在微生物生态学研究中逐渐得到了广泛的应用。从微生物生态学角度,综述了实时荧光定量PCR技术的原理、发展、优缺点及其在微生物生态学研究中的应用与研究进展,并探讨了实时荧光定量PCR技术的发展和应用前景。

用改进的重叠PCR引入血管内皮生长因子基因突变

血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)的PCR产物克隆于T载体上 ,经转化JM1 0 9感受态菌株后 ,随机挑取 8个白斑菌落 ,混合后制成混合模板 .采用 3条引物 ,做两轮重叠PCR反应 ,获得了VEGF的突变基因 ,经PCR鉴定 ,酶切鉴定和测序分析表明所得基因为目的产物 .实践证明这种突变方法简单快速 ,为下一步实验大量引入突变奠定了实验基础

Multiplex binding modes of toluidine blue with calf thymus DNA and conformational transition of DNA revealed by spectroscopic studies

It is noteworthy to understand the details of interactions between antitumor drugs and DNA because the binding modes and affinities affect their antitumor activities. Here, The interaction of toluidine blue (TB), a potential antitumor drug for photodynamic therapy of tumor, with calf thymus DNA (ctDNA) was explored by UV-vis, fluorescence, circular dichroism (CD) spectroscopy, UV-rnelting method and surface-enhance Raman spectroscopy (SERS). The experimental results suggest that TB could bind to ctDNA via both electrostatic interaction and partial intercalation.

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a "U" Re expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse "U" with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondria 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitocliondrial 12S rRNA gene, which were about 450 bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR amplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, L. erythopterus from L. argentintaculatus, L. malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for differentiation of L. sattguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The seminested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Published by Elsevier Ltd.