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Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

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Science china(Life Sciences) 2009年第5期52卷 483-491页

作者： SU YeYang1,2,3. LIN Lin1,2,3. TIAN Geng1,2,3. CHEN Chen2,3. LIU Tao2,3. XU Xingya3. QI XinPeng1,2,3. ZHANG XiuQing2,3. YANG HuanMing2,3.1 Graduate School of Chinese Academy of Sciences, beijing 100049, china 2 beijing Institute of {3., Chinese Academy of Sciences, beijing 100029, china 3.beijing {3. Institute, beijing 1013.0, china Graduate School of Chinese Academy of Sciences Beijing China beijing Institute of genomics Chinese Academy of Sciences Beijing China beijing genomics Institute Beijing China

To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3.3.xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) o

关键词： health genomics the next-generation sequencing techonology sequencing technology large-scale genomics PCR

Identification of human dopamine receptors agonists from Chinese herbs

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Acta Pharmacologica Sinica 2007年第1期28卷 132-139页

作者： Yi-lin ZHANG~(1,2,3.4) Hai-qing ZHANG~(1,2,3.4) Xiao-yu LIU~3.Shi-neng HUA~3.Lu-bing ZHOU~3.Jun YU~2 Xue-hai TAN~(3.5)1 Graduate School of the Chinese Academy of Sciences,beijing 10003.,china 2 beijing {3. Institute,Chinese Academy of Sciences beijing 1013.0,china 3.HD Biosciences Co Ltd,Shanghai 201203.china Graduate School of the Chinese Academy of Sciences Beijing China beijing genomics Institute Chinese Academy of Sciences Beijing China HD Biosciences Co Ltd Shanghai China

Aim:To find human dopamine receptors,especially Dl-like receptor specific ago-nists from Chinese herbs as potential antihypertension drug ***:TwoD l-like receptor cell lines carrying a μ-lactamase reporter gene,and a D2 receptorcell line coexpressing a promiscuous G protein G15 were constructed using HEK293.ells.A natural compound library made from fractionated samples of herbal ex-tracts was used for high-throughput screening（HTS）against one of the cell lines,HEK/D5R/*** interested hits were evaluated for their activities againstvarious dopamine ***:Fourteen hits were identified from primaryscreening,of which 2 of the better hit samples,HD0522 and HD0059,were selectedfor further material and activity analysis,and to obtain 2 compounds that ap-peared as 2 single peaks in HPLC,HD0522H01 and ***0059H01could activate D 1,D2,and D5 receptors,with EC50values of 2.28 μg/mL,0.85 μg/mL,and 1.41 μg/mL,***0522H01 could only activate DlR and D5Rwith EC50values of 2.95 μg/mL and 8.3. μg/***:We established cell-based assays for 3.different human dopamine receptors and identified specificagonists HD0522H01 and HD0059H01 through *** specific agonist to D1-like receptors,HD0522H01,may become a new natural product-based drug leadfor antihypertension treatment.

关键词： dopamine receptors natural products high throughput screening hypertension

Sequencing,annotation and comparative analysis of nine BACs of the giant panda(Ailuropoda melanoleuca)

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Science china(Life Sciences) 2010年第1期53卷 107-111页

作者： ZHENG Yang1,2,3.CAI Jing4,LI JianWen3.6,LI Bo3.LIN RunMao3.TIAN Feng3.WANG XiaoLing3.& WANG Jun3.5 1beijing Institute of genomics,Chinese Academy of Sciences(CAS),beijing 100029,{3. 2College of Life Science,Graduate University of Chinese Academy of Sciences,beijing 100049,{3. 3.0} genomics Institute at Shenzhen,Shenzhen 518083.{3. 4CAS-Max Planck Junior Research Group on Evolutionary genomics,State Key Laboratory of Genetic Resources and Evolution,Kunming Institute of Zoology,Chinese Academy of Sciences(CAS),Kunming 650223.{3. 5Department of Biology,University of Copenhagen,Copenhagen DK-2200,Denmark 6School of Biological Science and Engineering,South china University of Technology,Guangzhou 510006,{3. beijing Institute of genomics Chinese Academy of Sciences (CAS) Beijing China College of Life Science Graduate University of Chinese Academy of Sciences Beijing China beijing genomics Institute at Shenzhen Shenzhen China CAS-Max Planck Junior Research Group on Evolutionary genomics State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology Chinese Academy of Sciences (CAS) Kunming China School of Biological Science and Engineering South China University of Technology Guangzhou China Department of Biology University of Copenhagen Copenhagen Denmark

A 10-fold BAC library for the giant panda was constructed and nine BACs were selected to generate finish *** BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of the giant panda newly generated by Illumina GA sequencing *** Sanger sequencing,assembly,annotation and comparative analysis were carried out on the selected BACs of a joint length 878 *** search and de novo prediction methods were used to annotate genes and *** protein coding genes were predicted,seven of which could be functionally *** seven genes have an average gene size of about 41 kb,an average coding size of about 1.2 kb and an average exon number of 6 per ***,seven tRNA genes were *** 27 percent of the BAC sequence is composed of repeats.A phylogenetic tree was constructed using a neighbor-join algorithm across five species,including the giant panda,human,dog,cat and mouse,which reconfirms dog as the most closely related species to the giant *** results provide detailed sequence and structure information for new genes and repeats of the giant panda,which will be helpful for further studies about the giant panda.

关键词： giant panda BAC sequencing assembly annotation evolution

Recent progress in 8igenomic research of liver cancer

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Science china(Life Sciences) 2009年第1期52卷 24-30页

作者： HAN ZeGuang Shanghai-MOST Key Laboratory for disease and Health genomics,Chinese National human Genome C{3. at Shanghai,Shanghai 201203.china Shanghai-MOST Key Laboratory for disease and Health genomics Chinese National Human Genome Center at Shanghai Shanghai China

Along the course of occurrence and development of liver cancer,the corresponding somatic cells accumulate some important genetic *** variations may be divided into two *** the genetic changes closely related to etiology of liver cancer,the well-known cases include insertion and integration of the hepatitis B virus(HBV) DNA after infection,and mutations at site 249 of the tumor suppressor gene p53.induced by exposure to aflatoxin *** secondary genetic changes include amplification and deletion of certain chromosome regions,mutations in p53.at the sites other than 249,as well as the mutational activation of the Wnt/β-catenin signal *** tumor cells with these genetic variations may gradually become the dominant clones under evolutionary ***,identification of genetic susceptible against risk of liver malignancy is also an important aspect of research in this field.

关键词： hepatocellular carcinoma chromosomal instability gene mutation oncogene tumor suppressor gene

Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21,LipL3. and OmpL1 in Epidemic Leptospires

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 7+650-656页

作者： Xiang-Yan ZHANG~(1#) Yang YU~(2,3.) Ping HE~1 Yi-Xuan ZHANG~(2,3. Bao-Yu HU~1 Yang YANG~1 Yi-Xin NIE~4 Xiu-Gao JIANG~4 Guo-Ping ZHAO~(2,3. Xiao-Kui GUO~(1*) 1 Department of Medical Microbiology and Parasitology,Shanghai Jiaotong University,Shanghai 200025,china 2 Research Center of Biotechnology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 20003.,china 3.Chinese National {3. Genome Center at Shanghai,Zhangjiang High Tech Park,Shanghai 201203.china 4 Institute for Infectious disease Control and Prevention,Chinese Center for disease Control and Prevention,beijing 102206,china Department of Medical Microbiology and Parasitology Shanghai Jiaotong UniversityShanghai 200025 China Research Center of Biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Chinese National human Genome Center at Shanghai Zhangjiang High Tech ParkShanghai 201203 China Institute for Infectious disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206 China

Leptospiral outer membrane proteins (OMPs) are highly conserved in different species,andplay an essential role in the development of new immunoprotection and serodiagnosis *** genesencoding LipL21,LipL3. and OmpL1 were cloned from the complete genome sequence of Leptospirainterrogans serovar lai strain Lai and expressed in *** comparison analysis revealed that the threegenes were highly conserved among distinct epidemic leptospires,including three major epidemic speciesLeptospira interrogans,Leptospira borgpetersenii and Leptospira weilii,in *** analysis wasfurther performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the *** immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that thesethree OMPs were conservatively expressed in pathogenic *** strains and other pathogenic ***,the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA)for serodiagnosis of leptospirosis was *** recombinant LipL3. and OmpL1 proteins showed ahigh degree of ELISA reactivity with sera from patients infected with *** strain Lai and otherpathogenic *** results may contribute to the identification of candidates for broad-rangevaccines and immunodiagnostic antigens in further research.

关键词： outer membrane protein expression comparative analysis epidemic leptospire

Exploring in vitro roles of siRNA in cardiovascular disease

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Acta Pharmacologica Sinica 2007年第1期28卷 1-9页

作者： Yu TANG~(2,3. Yu-zhi GE~3.James Q YIN~(2,4)2 Institute of Biophysics,Chinese Academy of Sciences,beijing 100101,china 3.United Cardiovascular Department,Jiangxi Provincial People's Hospital,Nanchang 3.0046,china Institute of Biophysics Chinese Academy of Sciences Beijing China United Cardiovascular Department Jiangxi Provincial People's Hospital Nanchang China

RNA interference(RNAi)is an adaptive defense mechanism through which double-stranded RNAs silence cognate genes in a sequence-specific *** has beenemployed widely as a powerful tool in functional genomics studies,target valida-tion and therapeutic product ***,the application of small-interfering RNA(siRNA)to the silencing of the disease-causing genes involvedin cardiovascular diseases has made great *** this overview,we attemptto provide a brief outline of the current understanding of the mechanism of RNAiand its potential application to the cardiovascular system,with particular empha-sis on its ability to identify the pathophysiological function of genes related toseveral important cardiovascular *** prospects of RNAi-basedtherapeutics,as well as the advantages and potential problems,are also discussed.

关键词： siRNA RNAigene silencing cardiovascular disease gene therapy

Development and application of genotyping technologies

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Science china(Life Sciences) 2009年第1期52卷 17-23页

作者： SHI JinXiu,WANG Ying & HUANG Wei Shanghai-MOST Key Laboratory of Health and disease genomics,Chinese National human Genome C{3. at Shanghai,Shanghai 201203.china Shanghai-MOST Key Laboratory of Health and disease genomics Chinese National Human Genome Center at Shanghai Shanghai China

With the completion of human Genome Project,International HapMap Project and the publication of copy number variation in human genome,a great number of accurate,rapid,and cost-effective technologies for SNP analysis have been developed,promoting the research of the complex *** article presents a review of widely used genotyping techniques,and the progress and prospect in the study of complex diseases in terms of the projects and achievements of Chinese National human Genome Center at Shanghai(CHGCs).

关键词： single nucleotide polymorphism copy number variation complex disease

Identification and Characterization of glnA Promoter and its Corresponding Trans-regulatory Protein GlnR in the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U3.

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Acta Biochimica et Biophysica Sinica 2006年第12期38卷 831-843页

作者： Hao YU~(1&) Wen-Tao PENG~1 Yang LIU~1 Ting WU~2 Yu-Feng YAO~1 Ming-Xue CUI~1 Wei-Hong JIANG~1 Guo-Ping ZHAO~(1,3.4*) 1 Laboratory of Molecular Microbiology,Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 20003.,china 2 Department of Medicinal Chemistry,School of Pharmacy,Fudan University,Shanghai 20003.,china 3.State Key Laboratory of Genetic Engineering & Department of Microbiology,School of Life Science,Fudan University,Shanghai 20043.,china 4 Laboratory of {3. and Health genomics,Chinese National human Genome Center at Shanghai,Shanghai 201203.china Laboratory of Molecular Microbiology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China Department of Medicinal Chemistry School of Pharmacy Fudan University Shanghai 200032 China State Key Laboratory of Genetic Engineering & Department of Microbiology School of Life Science Fudan University Shanghai 200433 China Laboratory of disease and Health genomics Chinese National Human Genome Center at Shanghai Shanghai 201203 China

The genetic requirements for the transcription of glnA,encoding the major glutaminesynthetase in a rifamycin SV-producing Amycolatopsis mediterranei strain,U3.,were *** experiments showed that the promoter of U3. glnA （pglnA） was likely to have two transcriptioninitiation sites:P1and P2,located 157 and 45 nucleotides （nt） upstream of the translational start codon,*** mobility shift and DNase I footprinting analyses revealed a 3. bp cis-element locatedat 45 to 75 nt downstream of P1,or 3. to 68 nt upstream of *** sequence of the cis-element displayedhigh similarity to the corresponding regions of pglnA from Streptomyces coelicolor and *** as a reporter gene,the expression levels of U3. pglnA and its deletion derivatives under differentnitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in ***54,*** J508 and *** FS10 （glnR mutant）.These in vivo studies showed that theactivation of U3. pglnA in *** required GlnR,and its binding to the U3. pglnA was furtherconfirmed by the gel mobility shift *** and heterologous expression of the U3. glnR allowed usto detect the in vitro interaction between the U3. GlnR and the corresponding pglnA *** shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for theactive transcription of glnA in U3..

关键词： glnA promoter cis-element glnR Amycolatopsis mediterranei

Genomic research for important pathogenic bacteria in china

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Science china(Life Sciences) 2009年第1期52卷 50-63页

作者： YANG RuiFu1,GUO XiaoKui2,YANG Jian3.JIANG YongQiang1,PANG Bo4,CHEN Chen5,YAO YuFeng2,QIN JinHong2 & LI QingTian2 1 Laboratory of Analytical Microbiology,State Key Laboratory of Pathogen and Biosecurity,beijing Institute of Microbiology and Epidemiology,beijing 100071,china 2 Department of Medical Microbiology and Parasitology,Institutes of Medical Sciences,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,china 3.State Key Laboratory for Molecular Virology and Genetic engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,beijing 100176,china 4 State Key Laboratory for Infectious disease Prevention and Control,Institute for Communicable disease Control and Prevention,Chinese Center for disease Control and Prevention,beijing 102206,china 5 beijing {13. Institute at Shenzhen,Shenzhen 518083.china Laboratory of Analytical Microbiology State Key Laboratory of Pathogen and Biosecurity Beijing Institute of Microbiology and Epidemiology Beijing China Department of Medical Microbiology and Parasitology Institutes of Medical Sciences Shanghai Jiao Tong University School of Medicine Shanghai China State Key Laboratory for Molecular Virology and Genetic engineering Institute of Pathogen Biology Chinese Academy of Medical Sciences Beijing China State Key Laboratory for Infectious disease Prevention and Control Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing China beijing genomics Institute at Shenzhen Shenzhen China

Rapid accumulation of bacterial genomic data offered an unprecedented opportunity to understand bacterial biology from a holistic view of *** can thus closely look at the way in which a pathogen is evolved,and these data has been applied to molecular epidemiology and microbial forensics,and screening of novel diagnostic,vaccine and drug *** newly developed high-throughput low-cost sequencing technologies,such as 454,Solexa and SOLiD,will promote the acquisition and application of genomic data in new research areas that we dared not imagine previously,such as the metagenomics of human gastric-intestinal tract,for better and comprehensive understanding of human health and {3..

关键词： genome pathogenic bacteria sequencing application