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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 genomics 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

Discovery of immune related factors in Fenneropenaeus chinensis by annotation of ESTs

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Progress in Natural Science:Materials International 2004年第1期14卷 48-55页

作者： SHEN Yaoqing 1,2 , XIANG Jianhai 1*, WANG Bing 1,2 , LI Fuhua 1 and TONG Wei 3.1. Experimental Marine Biology Laboratory, institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071,China 2. Graduate School of the Chinese Academy of Sciences, beijing 10003., China 3. genomics & Bioinformatics institute, Chinese Academy of Sciences, beijing 1013.0, China) Chinese Acad Sci Inst Oceanol Expt Marine Biol Lab Qingdao 266071 Peoples R China Chinese Acad Sci Grad Sch Beijing 100039 Peoples R China Chinese Acad Sci Geonom & Bioinformat Inst Beijing 101300 Peoples R China

A total of 10446 expressed sequence tags (ESTs) are obtained by a large scale sequencing of a cDNA library from cephalothorax of adult Fenneropenaeus chinensis . An EST analysis platform was built up based on local computers and bioinformatic techniques were used to annotate these ESTs in order to promptly find possible functional genes, especially for immune related factors. About 4% of the ESTs show similarity to the coding sequences of such factors, including lectin, serine protease, serpin, lysozyme, etc. These ESTs provide a partial profile of the immune system in F. chinensis and useful information for further study on these genes.

关键词： Fenneropenaeus chinensis bioinformatics ESTs immune factors

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 genomics {3.,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

Expression of PDCD5 , a novel apoptosis related protein, in human osteoarthritic cartilage

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Acta Pharmacologica Sinica 2004年第5期25卷 143-148页

作者： Ai-xin CHENG, Si-quan LOU2, Hao-wei ZHOU, Ying WANG3. Da-long MA3.epartment of Orthopaedic Surgery, Third Hospital, Peking University, beijing 100083.3.enter for Human Disease genomics, Peking University, beijing 100083. China Department of Orthopaedic Surgery Third Hospital Peking University 北京 100083 Center for Human Disease genomics Peking University 北京 100083

AIM: To investigate the expression features of PDCD5 (programmed cell death 5 protein) in osteoarthritic andnormal human cartilage, and speculate on its potential functions in the pathogenesis of osteoarthritis (OA).METHODS: Articular cartilage specimens were obtained from 3. patients with OA and 16 healthy patients at thetime of arthroplasty. Expression of PDCD5 was detected by flow cytometry, immunofluorescence, RT-PCR andimmunohistochemical analysis. RESULTS: Enhanced expression and nuclear accumulation of PDCD5 in OAchondrocytes were found. PDCD5-positive chondrocytes were mainly distributed in the superficial and deep zonesof OA tissue sections, as opposed to, in the superficial and middle regions of normal healthy tissue ***: Since apoptotic chondrocyte death occurs more frequently in OA cartilage than in normal healthycartilage and PDCD5 is an apoptosis-related protein, the different expression patterns of PDCD5 in OA cartilagefrom that in normal healthy cartilage indicate that PDCD5 is involved in the pathogenesis of OA.

关键词： apoptosis cartilage osteoarthritis PDCD5

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro1

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Acta Pharmacologica Sinica 2005年第10期26卷 1274-1280页

作者： Shu-jia DAI~2, Gui-fang DOU~3. Xiao-hong QIAN~(4,5), Hai-feng SONG~4, Zhong-ming TANG~2, De-sheng LIU~2, Xiu-wen LIU~2, Liu-meng YANG~6, Yong-tang ZHENG~6, Qing LIANG~7 ~2 Laboratory of Metabolism of Biotechnology Derived Drugs, beijing institute of Radiation Medicine, beijing 100850 ~3.Laboratory of Drug Metabolism and Pharmacokinetics, beijing institute of Transfusion Medicine, beijing 100850 ~5 Laboratories of genomics and Proteomics, beijing institute of Radiation Medicine, beijing 100850 ~6 Laboratory of Molecular hnmunopharmacology, Kunming institute of Zoology, Chinese Academy of Sciences, Kunming 650223.~7 FusoGen Pharmaceuticals Inc, Tianjin 3.0051, China Laboratory of Metabolism of Biotechnology Derived Drugs Beijing Institute of Radiation Medicine Beijing China Laboratory of Drug Metabolism and Pharmacokinetics Beijing Institute of Transfusion Medicine Beijing China Laboratories of genomics and Proteomics Beijing Institute of Radiation Medicine Beijing China Laboratory of Molecular Immunopharmacology Kunming Institute of Zoology Chinese Academy of Sciences Kunming China FusoGen Pharmaceuticals Inc Tianjin China

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus （HIV） peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry （SPELC/MS/MS） was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-127I iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000μg/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10 626±2 886μg/L and 528±191μg/L, and the terminal imination half-lives (T1/2) were 6.3.0.9 h and 5.5±1.0 h, respectively. After sc, Tmaxwas 0.25-2h, and the absolute bioavailability was 49%±13.. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50of 0.3.μg/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T1/2of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50in vitro.

关键词： sifuvirtide pharmacokinetics HIV HIV fusion inhibitors enfuvirtide on-line solid-phase extraction macaque

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,China 2College of Life Science,Graduate University of Chinese Academy of Sciences,beijing 100049,China 3.0} genomics institute at Shenzhen,Shenzhen 518083.China 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.China 5Department of Biology,University of Copenhagen,Copenhagen DK-2200,Denmark 6School of Biological Science and Engineering,South China University of Technology,Guangzhou 510006,China 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

Sequencing and analysis of the giant panda genome

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Science China(Life Sciences) 2010年第8期53卷 1047-1048页

作者： YANG HuanMing BGI-Shenzhen (beijing genomics institute Shenzhen), Shenzhen 518083. China BGI-Shenzhen (beijing genomics institute Shenzhen) Shenzhen China

The giant panda (Ailuropoda melanoleuca) is loved all over the world and is considered a symbol of China, as illustrated by its being one of the

关键词： Sequencing and analysis of the giant panda genome

High Throughput SNP Genotyping with Two Mini-sequencing Assays

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 379-384页

作者： Chunqing LUO1,2#, Libin DENG1#, and Changqing ZENG1,2* The institute of Genetic and Developmental Biology, the Chinese Academy of Sciences, beijing 100101, China 2beijing {3. institute, the Chinese Academy of Sciences, beijing 1013.0, China The institute of Genetic and Developmental Biology the Chinese Academy of SciencesBeijing 100101 China beijing genomics institute the Chinese Academy of SciencesBeijing 101300 China

Two mini-sequencing methods, FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry), were optimized. A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay, thus made the optimization work easier. At the same time, using multi-PCR technology, 8-plex genotyping of MassArray assay was successfully carried out, some softwares were developed and the data process of MassArray was highly automated. Then these two methods were applied to high throughput SNP genotyping, the accuracy, efficiency and robustness were compared. The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product, as well as extension cycles, the SNPprimer length of FP-TDI should be 24–3. bp long, whereas MassArray assay prefers to be as short as only 16 *** 6440 SNP sites of human chromosome 3.were genotyped in a sample of 90 individuals, 4792 sites by FP-TDI assay and 1648 sites by MassArray assay, the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively. The throughput of MassArray was higher than FP-TDI, and the cost of MassArray was lower, MassArray was more suitable for high throughput SNP genotyping.

关键词： SNP genotyping FP-TDI MassArray high throughput

A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 817-823页

作者： Qing-Ming WANG*, Guo-Cai FAN, Ji-Zhong CHEN, Hui-Peng CHEN, and Fu-Chu HE* Department of genomics and Proteomics, beijing institute of Radiation Medicine, beijing 100850, China Department of genomics and Proteomics Beijing Institute of Radiation MedicineBeijing 100850 China

Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mecha- nisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

关键词： Apoptin nuclear localization signal cytoplasmic retention signal nuclear export signal