Retrieval and Amplification of DNA from Unstained Histopathological Sections

Testing of compounds for carcinogenic potential in vivo involves various experimental designs. A few of these techniques are directed to demonstrate the genotoxicity and mutagenicity of the compound by histopathology. These changes shown by histochemical means include monoclonal antibody directed cellular markers. Development of the polymerase chain reaction technique (PCR) for amplification of DNA has facilitated the investigation of molecular events related to the formation of malignant neoplasms. We describe here a method for screening tissues for mutations of the H-ras gene using monoclonal antibodies directed toward normal and mutant p21 proteins. Formalin-fixed, paraffin-embedded tissue sections are used to subsequently confirm the gene mutation by PCR amplification of the H-ras gene. The results indicated a successful application of this technique to demonstrate the presence of p21 oncoprotein in the tissues tested.

Primers for the Amplification of the Circular Chloroplast DNA from the A-genome Group of Cultivated Cotton

The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application

DETECTION AND APPLICATION OF MICROFLUIDIC ISOTHERMAL AMPLIFICATION ON CHIP

Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method.Compared with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures.In this paper,we designed and fabricated different kinds of polycarbonate(PC)microfluid chips,explored appropriate reaction condition for LAMP in microenvironment(1 nL→10μL),and developed a microfluidic isothermal amplification detection system.The DNA optimal amplification temperature is obtained;the starting time of exponential amplification of DNA is put forward farther.The optimal condition of DNA amplification in microenvironment,with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

Analysis of DNA methylation in different tissues of Fenneropenaeus chinensis from the wild population and Huanghai No. 1

DNA methylation plays an important role in the regulation of gene expression during biological development and tissue differentiation in eukaryotes. A methylation sensitive amplification polymorphism（MSAP） including digestion, pre-selective amplification and selective amplification was optimized to compare the levels of DNA cytosine methylation at CCGG sites in muscle, gill and hemocyte from the wild populations and the selective breeding of Huanghai No. 1 of Fenneropenaeus chinensis, respectively. Significant differences in cytosine methylation levels among three tissues in two populations were detected. The average DNA methylation ratios in muscle, gill and hemocyte of the wild population were 23.1%, 22.3% and 19.7%, while those were 21.4%, 19.6%,and 18.9% in Huanghai No. 1, respectively. The DNA methylation levels of gill from the two populations were highly significant（P〈0.01）, the difference of muscle was significant（P〈0.05）, while in hemocyte, there were no significant differences（P〉0.05）. DNA polymorphic methylation of gill and hemocyte between the wild population and Huanghai No. 1 varies to some extent, while those of muscle kept in a balanced degree. Furthermore,polymorphic methylation was associated with demethylation and methylation of CCGG loci.

Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR

[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.

An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles（MALBAC） kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although Genome Plex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content.

Molecular Cloning and Preliminary Analysis of a Fragile Site Associated Gene

Objective To analyze the molecular coining of a fragile site-associated gene. Methods Genomic Chinese hamster ovary （CHO） DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions （RT-PCR）. The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues. Results To investigate the molecular mechanism underlying the initial events of mdrla amplification, we cloned lq31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated （FSA） gene. FSA encoded an unusually large mRNA of - 16 kb. Full-length human FSA eDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder. Conclusion FSA plays an important role in regulating mammalian epithelial cell growth and differentiation.

基于SCAR标记和DNA条形码技术的苍术基原鉴别研究

目的开发出能同时鉴别北苍术和关苍术的分子标记方法,并探究不同种质资源苍术的遗传进化关系。方法对不同地区北苍术Atractylodes chinensis(Bunge)Koidz及关苍术A.japonica Koidz.ex Kitam基因组DNA的差异片段进行测序,结合SRAP、ISSR、DAMD分子标记方法,优化PCR反应体系,筛选并转换成特异性标记,同时,采用条形码方法分析种间序列差异。结果通过SRAP、ISSR、DAMD三种分子标记方法的PCR扩增,共筛选出198对能稳定扩增且重现性好的引物,转换出7对能稳定、快速鉴别北苍术和关苍术的SCAR引物。条形码方法检测出北苍术ITS2序列长度为454 bp,关苍术ITS2序列长度为453 bp,与其他苍术属植物之间遗传距离较远。NJ树结果显示,北苍术、关苍术及其他苍术属植物均各自聚为一支,表现出良好的单系性。依据ITS2二级结构,4种苍术属植物在螺旋区的茎环数目、大小、位置均有明显差异,可以直观地进行区分。结论所开发的特异性SCAR标记为苍术属植物优良品种的筛选提供了新方法,DNA条形码能稳定、准确鉴别北苍术。

Characterization of Sporothrix schenckii by random amplification of polymorphic DNA assay

s To investigate the DNA polymorphism of Sporothrix schenckii ( S schencki i ) and to find the relationship between DNA patterns and geographic areas and clinical manifestations Method The total DNA was extracted with hexadecyltrimethyl ammonium bromide Random A mplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S schenckii collected from different areas and isolated from di fferent clinical types Results Of seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5' ACCCGACCTG 3', 5' GAGAGCCAAC 3', 5 ' GGTGAC^GCAG 3' respectively The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability Differ ent isolates showed a common conserved DNA band with the same primer Different clinical types showed different genotypes Conclusion RAPD analysis is useful in DNA typing of S schenckii , the DNA band type of which is related to geographic origin and Clinical manifestation

Identification RAPD Markers Linked to Texture Quality of Chinese Cabbage

[ Objectives] This study was conducted to identify the random amplification of polymorphic DNA （RAPD） markers linked to chewy texture-controlling gene of Chinese cabbage. [ Methods] The RAPD markers associated with chewy texture of Chinese cabbage were identified via bulked segregant analysis （BSA） in an F2 population derived from the cross between Hua 273 （female parent） and 114 Fushan （male parent）. [ Results] OPA06-1400 was identified to he linked to the chewy texture-controlling gene of Chinese cabbage. The genetic distance between the target gene and the RAPD marker was 24.8 cM. [ Conclusions] The resuits provide experimental evidence for breeding of Chinese cabbage.

DNA Nanotechnology for Multimodal Synergistic Theranostics

Over the past decade,DNA nanotechnology has developed rapidly due to its unique characteristics,such as excellent biocompatibility,high programmability,good predictability,automatically chemical synthesis,and so on.So far,a variety of DNA-based nanostructures,from small to large and simple to complex,have been designed and synthesized with controllable size and shape in one,two,or three dimensions.Therefore,DNA has become a kind of competitive materials for biosensing,bioimaging and biomedicine.In particular,the integration of DNA nanotechnology with multimodal synergistic theranostics can not only achieve accurate cancer diagnosis by the sensitive and accurate detection of cancer biomarkers,but also achieve enhanced anti-cancer therapeutic efficacy,which promote the development of DNA nanotechnology and nanomedicine.In this review,we first give a comprehensive introduction of DNA nanotechnology,and then summarize the DNA self-assembly and amplification strategies for the construction of functional nanoplatforms for multimodal synergistic theranostics.Finally,the challenges and opportunities faced by DNA nanotechnology in biomedicine are discussed.

Loop-mediated isothermal amplification of single pollen grains

The polymerase chain reaction （PCR） has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental require- ments of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplifica- tion methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop- mediated DNA amplification method （LAMP）, an isothermal DNA amplification technique, to be used in pollen analysis. Wedemonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.