王进科
主要从事DNA结合蛋白(特别是序列特异性转录因子)新型高通量、高灵敏性检测分析技术及生物信息获取技术的发展及应用研究。
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- 姓名:王进科
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学术头衔:
博士生导师, 教育部“新世纪优秀人才支持计划”入选者
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学科领域:
物理海洋学
- 研究兴趣:主要从事DNA结合蛋白(特别是序列特异性转录因子)新型高通量、高灵敏性检测分析技术及生物信息获取技术的发展及应用研究。
王进科,男,1969年12月生,博士,现为东南大学生物科学及医学工程学院、生物电子学国家重点实验室教授,博士研究生导师。
主要从事DNA结合蛋白(特别是序列特异性转录因子)新型高通量、高灵敏性检测分析技术及生物信息获取技术的发展及应用研究,如双链DNA微阵列(dsDNA microarray)芯片的制备技术及应用研究、DNA结合蛋白(DNA-binding protein)的检测技术及应用研究、DNA/蛋白质相互作用(DNA/protein interaction)的分析技术及应用研究、基因表达调控的通路及网络(pathway and network of gene transcription regulation)的构建技术及应用研究、转录因子活性调控药物的筛选技术及应用研究等。
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成果数
12
【期刊论文】A novel method for screening species-specific gDNA probes for species identification
王进科, Tong Xiang Li, JinKe Wang, YunFei Bai, XiangDong Sun and ZuHong Lu*
Nucleic Acids Research 32 4 (2004): 1-8,-0001,():
-1年11月30日
We report a method called SSH array which combines the suppression subtraction hybridization (SSH) and DNA array techniques to find species specific DNA probes from genomic DNA (gDNA) for species identification. The method first obtains the differential gDNA fragments between two species by SSH and then hybridizes the differential gDNA fragments with arrays made of multiple whole genomes from several species to screen the unique gDNA fragments for one species. The screened unique gDNA fragments can be used as species specific probes to differentiate the species they represent from all other species. We used five species of the genus Dendrobrium, D.aurantiacum Kerr, D.officinale Kimura et Migo, D.nobile Lindl., D.chrysotoxum Lindl. and D.fimbriatum Hook., as experimental materials to study the feasibility of the method. The results showed that the method could efficiently obtain different species-specific probes for each of the five species.
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【期刊论文】Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins
王进科, Jinke Wang*, Tongxiang Li, Xiaoying Guo and Zuhong Lu
Nucleic Acids Research 33 2 (2005): 1-9,-0001,():
-1年11月30日
We describe a new method for the assay of sequencespecific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNAfootprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNAbinding proteins. We named this method as exonuclease III(ExoIII) protectionassaywithFRETprobe.The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion,resulting in high FRET. However, if the target protein does not exist, theFRETpaironthe nakedFRETprobe will be degraded by ExoIII, resulting in low FRET.Three kinds of recombinant transcription factors including NF-kB, SP1 and p50, and the target protein of NF-kB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.
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王进科, Jinke Wang a, b, Yunfei Bai a, Tongxiang Li a, Zuhong Lu a, *
J. Biochem. Biophys. Methods 55 (2003): 215-232,-0001,():
-1年11月30日
We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3Vhydroxyl end and an overhang sequence at 5Vamino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3V end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-nB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.
DNA microarrays, Double-stranded DNA probes, DNA/, protein interactions
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【期刊论文】A method for fabricating uni-dsDNA microarrayn chip for analyzing DNA-binding proteins
王进科, Jin K. Wang*, Tong X. Li, Zu H. Lu
J. Biochem. Biophys. Methods 63 (2005): 100-110,-0001,():
-1年11月30日
This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5V end were firstly annealed to a same universal oligonucleotide with amino group at 5V end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3V end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins. D 2005 Elsevier B.V. All rights reserved.
Fabrication, uni-dsDNA microarray, DNA-binding protein
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王进科, Jinke Wang, Min L. Li, Dong Hua, and Qixin Chen
,-0001,():
-1年11月30日
This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor κB (NF-κB). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT, contained a NF-κB binding consensus sequence for capturing activated NF-κB in analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-κB. Finally, the probes protected by NF-κB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-κB without the need for NF-κB antibodies. EMEA may provide a general approach for assays of DNA sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.
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王进科, Jinke Wang, , Yunfei Bai, Tongxiang Li, Nongyue He, Zuhong Lu*
Advanced Nanomaterials and Nanodevices IUMRS-ICEM 2002, Xi'an, China, 10-14 June 2002,-0001,():
-1年11月30日
The paper manufactured the short double-stranded DNA (dsDNA) arrays on glass surface for detecting sequence-specific interactions of immobilized dsDNA with transcription factors in sample. The short dsDNA arrays were fabricated by firstly immobilizing the amino-linked single-stranded oligonucleotides as short as 15~26bp onto glutaraldehyde-derived glass slides, and subsequently annealing with the complementary single-stranded oligonucleotides. The arrayed dsDNAs harbored dsDNA-binding sites of certain transcription factors. The fabricated dsDNA arrays were respectively hybridized with transcription factor as NF-kB, AP1 or AP2, and HeLa nuclear extract. It is demonstrated that the arrayed short dsDNA is accessible to transcription factors in samples, and the interactions of dsDNA with target transcription factors are strongly sequence-specific. It is also revealed that the arrayed short dsDNA can sensitively detect the target transcription factors as low as 5ng/mL in samples. We hypothesize that such a short dsDNA array on glass surface has great potential to be an excellent platform for parallel detection of expression of transcription factors in cells and the sequence-specific interactions of dsDNA with transcription factors.
double-stranded DNA array, trans, c, r, i, p, t, ion factors
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王进科, Jinke Wang, Tongxiang Li, Yunfei Bai, Yi Zhu and Zuhong Lu*
Molecules 8 (2003): 153-168,-0001,():
-1年11月30日
We present a novel method for fabricating unimole cular double-stranded DNA microarrays on solid surfaces, which were used to probe sequence-specific DNA/protein interactions. For manufacturing the unimolecular double-stranded DNA microarrays, two kinds of special single-stranded oligonucleotides, constant oligonucleotide and target oligonucleotide, were chemically synthesized. The constant oligonucleotides with internal aminated dT were used to capture and immobilize the target oligonucleotides onto the solid surface, and also to provide a primer for later enzymatic extension reactions, while target oligonucleotides took the role of harbouring DNA-binding sites of DNA-binding proteins. The variant target oligonucleotides were annealed and ligated with the constant oligonucleotides to form the new unimolecular oligonucleotides for microspotting. The prepared unimolecular oligonucleotides were microspotted on aldehyde-derivatized glass slides to make partial-dsDNA microarrays. Finally, the partial-dsDNA microarrays were converted into a unimolecular complete-dsDNA microarray by a DNA polymerase extension reaction. The efficiency and accuracy of the polymerase synthesis were demonstrated by the fluorescent-labeled dUTP incorporation in the enzymatic extension reaction and the restriction endonuclease digestion of the fabricated unimolecular complete-dsDNA microarray. The accessibility and specificity of the sequence-specific DNA-binding proteins binding to the immobilized unimolecular dsDNA probes were demonstrated by the binding of Cy3 labeled NF-?B (p50
Fabrication, Unimolecular double-stranded DNA microarray, enzyme extension, DNA-protein interactions
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62浏览
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王进科, Tongxiang Li, Jinke Wang, Zuhong Lu*
J. Biochem. Biophys. Methods 62 (2005): 111-123,-0001,():
-1年11月30日
About 63 species of Dendrobium are identified in China, making the identification of the origin of a particular Dendrobium species on the consumer market very difficult. We report evaluation of multiple species-specific probes screened from genomic DNA for closely related Dendrobium species identification, based on DNA array hybridization. Fourteen species-specific probes were screened from five closely related Dendrobium species, D. aurantiacum Kerr, D. officinale Kimura et Migo, D. nobile Lindl., D. chrysotoxum Lindl. and D. fimbriatum Hook., based on the SSH-Array technology we developed. Various commercial Dendrobium samples and unrelated samples were definitely identified. The specificity and accuracy of the multiple species-specific probes for species identification was assessed by identifying various commercial Dendrobium samples (Herba Dendrobii). Hybridization patterns of these multiple probes on digested genomic DNAs of Dendrobium species indicated that there are distinct polymorphic sequence fragment in the higher eukaryotes. This is the first report on detection and utilization of multiple speciesspecific probes of Dendrobium in whole genomic DNA, and this could be useful tools not only for a new technical platform for the closely related species identification but also for epidemiological studies on higher eukaryotes.
Identification, DNA array, Species-specific probes, Dendrobium
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【期刊论文】Optimization of on-chip elongation for fabricating double-stranded DNA microarrays
王进科, Yunfei Bai, Qinyu Ge, Jinke Wang, Tongxiang Li, Quanjun Liu, Zuhong Lu∗
Colloids and Surfaces B: Biointerfaces 40 (2005): 153-158,-0001,():
-1年11月30日
The sequence-specific recognitions betweenDNAand proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays; (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extensionwas about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50℃ and the Mg2+ concentration of 2.5mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.
dsDNA microarrays, Taq DNA polymerase, On-chip elongation, DNA-protein interactions
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王进科, Yun Fei BAI, Qin Yu GE, Tong Xiang LI, Jin Ke WANG, Quan Jun LIU, Zu Hong LU∗
Chinese Chemical Letters 16 5 (2005): 651-654,-0001,():
-1年11月30日
The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA-inding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.
Double stranded DNA microarray, DNA binding protein, label-free detection.,
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