线粒体多态性检测寡核苷酸芯片的构建

目的构建用于线粒体DNA（mtDNA）D-环控制区（D—LOOP区）多态性多个位点集成检测的寡核苷酸芯片。方法根据GenBank中人mtDNA D—LOOP区序列设计2组引物[普通引物和N,N＇-对羧苄基吲哚三菁（Cy5）标记引物]和55种寡核苷酸探针。将探针固定于醛基修饰载玻片表面,制备mtDNA多态性检测芯片。提取40例健康人外周血标本mtDNA,应用Cy5标记引物不对称PCR扩增mtDNA D—LOOP区片段。取未变性和变性后扩增产物分别与芯片进行杂交,观察二者杂交信号强度差异,并将芯片检测结果与DNA测序结果进行比较。观察等倍稀释的不对称PCR扩增产物的杂交信号强度,检测芯片的灵敏度;将DNA测序结果与探针进行BLAST2比对,找出与PCR扩增产物完全匹配和仅1个碱基不匹配的探针,分析二者在芯片上相应位点杂交信号强度的差异,观察芯片的特异性;以放置6个月后的芯片检测外周血mtDNA,观察杂交信号强度的变化。结果不对称PCR扩增获得的mtDNA D—LOOP区片段（466bp）与预期表达片段大小一致。不对称PCR扩增产物直接杂交和变性后杂交的信号强度无明显差异,40例健康人外周血标本mtDNA的芯片杂交检测结果均与DNA测序结果完全吻合。灵敏度检测结果显示,杂交信号强度随扩增产物稀释程度的增加而减弱,芯片检测靶分子的灵敏度为0．1ng;与DNA测序结果完全匹配的探针174和仅相差1个碱基“C”的探针174c的杂交信号强度具有明显差异;而保存6个月后的芯片杂交信号强度基本保持不变。结论构建的寡核苷酸芯片可满足对mtDNA D—LOOP区多态性进行多个位点快速通量检测的需要,具有较高的灵敏度和特异性,适用于法医鉴定及线粒体疾病的检测。     

肠道病毒71型60mer寡核苷酸探针的设计

目的设计肠道病毒71型（EV71）诊断芯片的寡核苷酸探针。方法使用NCBI获取全基因组序列,利用BLAST系统对基因序列进行比对,获取特异性序列;利用Array designer4．2对其进行寡核苷酸探针设计。结果设计出12条60mer寡核苷酸探针,为芯片的制备做准备。结论将BLAST系统和Array designer4．2软件相结合,能快速有效地设计出诊断芯片的探针.     

合成肽库及其应用

合成肽库由含特定长度的肽片段所组成，它包含了该长度短肽的各种可能序列或绝大部分序列。合成肽库家族包括随机合成肽库、合成肽组合文库（SPCL）、多用途肽库（MUPL）、位置扫描合成肽组合文库（PS．SPCL）、寡核苷酸编码的合成肽库等。肽库技术除可用于蛋白质研究及分子识别，也开始应用于免疫学领域，如抗原表位分析、药物设计及疫苗研制等方面。     

合成肽库及其应用

合成肽库由含特定长度的肽片段所组成，它包含了该长度短肽的各种可能序列或绝大部分序列。合成肽库家族包括随机合成肽库、合成肽组合文库（ＳＰＣＬ）、多用途肽库（ＭＵＰＬ）、位置扫描合成肽组合文库（ＰＳ－ＳＰＣＬ）、寡核苷酸编码的合成肽库等。肽库技术除可用于蛋白质研究及分子识别，也开始应用于免疫学领域，如抗原表位分析、药物设计及疫苗研制等方面。     

HIV-1基因限制性显示片段的制备（英文）

目的　快速分离HIV 1基因片段制备DNA芯片探针。　方法　以Sau3AⅠ酶切HIV基因后 ,将得到的限制性酶切片段两端接上接头。根据酶切位点与接头的序列设计通用引物。在该通用引物的 3’端分别延伸 1个碱基后 ,通过引物间的两两组合 ,将PCR反应分成 10个亚组。纯化各组PCR产物 ,克隆到T载体上。挑取白色菌斑进行快速鉴定后扩大培养阳性克隆、提质粒。以质粒为模板扩增靶片段并测序。　结果　每个亚型得到了十几个 10 0～ 10 0 0bp的HIV基因片段。　结论　限制性显示技术是一种有效的快速分离制备基因片段的方法。     

IGF—IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡的病理学研究

目的：探讨IGF-IR基因的反义硫代磷酸型寡核苷酸对胶质瘤细胞的形态学影响。方法：根据IGF-IRcDNA序列设计正义,反义寡核苷酸片段,并对其部分碱基进行硫代磷酸修饰。体外培养的胶质瘤细胞分别经正义寡核苷酸和反义寡核苷酸处理,应用倒置显微镜活细胞观察,HE染色光镜观察,透射电镜及DNA琼脂糖凝胶电泳等方法研究IGF-IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡作用。结果：经反义寡核苷酸转染的胶质瘤细胞中,呈现典型的凋亡形态学改变,凋亡细胞最早期表现为细胞体积,容量减少,染色质凝聚,继之染色质集聚于核膜下成7新月状或块状;细胞核内染色质可完全固缩成团呈“黑洞”样或萎缩的核破裂形成一些较小的膜包绕的球体位于胞质内,最后出泡形成凋亡小体,DNA琼脂糖凝胶电泳分析经反义寡核苷酸处理的胶质瘤细胞DNA降解片段,可见有明显的小分子量DNA梯状条带,而野生型和正义寡核苷酸处理的胶质瘤细胞未见DNA梯状条带。结论：IGF-IR所介导失发泌环路IGF-I/IGF-IR。在胶质瘤细胞增殖和维持恶性表型中起重要作用。IGF-IR反义硫代磷酸型寡核苷酸能诱导胶质瘤细胞凋亡。     

流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。     

人4-1BBL基因重组腺病毒载体的构建及其在HEK293细胞的表达

目的 体外构建含有人4-IBBL基因的重组腺病毒载体,并检测其在HEK293细胞中的表达.方法 设计一对含有Sfil酶切位点的4-1BBL因上下游引物,以质粒pCR4-TOP0-4-1BBL为模板,通过PCR扩增获得4-1BBL基因全序列.片段回收后经酶切处理,连接到穿梭质粒pShuttle-CMV-EGFP上,获得重...     

cDNA微阵列与寡核苷酸芯片的制备方法

c DNA微阵列和寡核苷酸芯片是常见的合成后点样的 DNA微阵列 ,点样方法主要是通过物理吸附或共价结合的方式将探针固定于载体上。本文总结了近年来国内外文献报道的 c DNA微阵列制备方法 :在多聚赖氨酸包被的玻璃基片表面制备 c DNA微阵列 ;用琼脂糖包被的玻璃基片制备 c DNA微阵列 ;在氨基或醛基修饰的玻璃基片表面制备 c DNA微阵列 ;寡核苷酸芯片的制备方法 :氨基修饰的玻片与 5 `末端带氨基的寡核苷酸探针通过不同的 linker连接 ;硅烷化寡核苷酸直接点样于玻片上制成寡核苷酸微阵列 ;硫代寡核苷酸通过二硫键与巯基修饰的玻片连接 ;水凝胶芯片固定寡核苷酸 ;丙烯酰胺硅烷化的基片与 5′丙烯酰胺修饰的寡核苷酸连接。并展望了基因芯片的应用前景     

70 mer长寡核苷酸探针芯片制备、标记方法及杂交条件的优化

目的优化长片段寡核苷酸芯片制备、探针标记及杂交条件。方法通过直接标记方法优化芯片的点样浓度、点样后的固定方法。探索两种靶基因标记方法,并优化杂交液及各种杂交条件。结果采用70mer寡核苷酸探针,点样浓度在10μmol/L时就可获得满意的荧光强度。探针点样后37℃水合,然后在3600mJ条件下进行紫外交联固定效果最好。样品采用生物素间接标记较CY3直接标记信号强度强,成本相对低廉。优化出的杂交液成分:50%甲酰胺、5×SSC、0.5%SDS,杂交时间为8h,杂交温度为65℃。结论通过此试验优化了制备病原体长探针寡核苷酸芯片的制备方法,样品标记策略和杂交液及杂交温度,为下一步制备多种病原体的寡核苷酸芯片检测系统奠定了基础。     

Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers.

A new procedure for sequence-independent PCR amplification of DNA fragments is described. DNA from pUC18 plasmid was used as a test DNA. It was digested with a frequently cutting restriction enzyme (Sau3A), generating sticky ends. The DNA was ligated to a synthetic, non-phosphorylated adaptor and subsequently amplified in a nested PCR using two oligonucleotides with sequences derived from the adaptor. As little as 1 fg of pUC18 DNA could be detected by this procedure. The product was analyzed on a gel and hybridized with a pUC18-specific probe. The sequence-independent nested PCR was repeated with different amounts of pUC18 DNA in the presence of an excess of non-specific DNA. In these experiments, pUC18 DNA fragments were amplified in a concentration-dependent manner. After hybridization with a digoxigenin dUTP-labelled pUC18 DNA probe, 1 fg of pUC18 DNA could still be detected. This method allows rapid screening of blood for low titred and mutated viruses in which primer binding sites are not conserved.     

Estimated number of off‐target candidate sites for antisense oligonucleotides in human mRNA sequences

Antisense oligonucleotide (ASO) therapeutics are single‐stranded oligonucleotides which bind to RNA through sequence‐specific Watson–Crick base pairings. A unique mechanism of toxicity for ASOs is hybridization‐dependent off‐target effects that can potentially occur due to the binding of ASOs to complementary regions of unintended RNAs. To reduce the off‐target effects of ASOs, it would be useful to know the approximate number of complementary regions of ASOs, or off‐target candidate sites of ASOs, of a given oligonucleotide length and complementarity with their target RNAs. However, the theoretical number of complementary regions with mismatches has not been reported to date. In this study, we estimated the general number of complementary regions of ASOs with mismatches in human mRNA sequences by mathematical calculation and in silico analysis using several thousand hypothetical ASOs. By comparing the theoretical number of complementary regions estimated by mathematical calculation to the actual number obtained by in silico analysis, we found that the number of complementary regions of ASOs could be broadly estimated by the theoretical number calculated mathematically. Our analysis showed that the number of complementary regions increases dramatically as the number of tolerated mismatches increases, highlighting the need for expression analysis of such genes to assess the safety of ASOs.     

Preferential recognition of specific DNA motifs by anti-double-stranded DNA autoantibodies

Although antibodies (Ab) specific for double-stranded (ds) DNA are thought to be involved in the etiopathogenesis of systemic lupus erythematosus (SLE), the fine structure of their DNA targets remains elusive. We have adapted a polymerase chain reaction (PCR)-assisted immunoprecipitation method to define the binding sites in DNA sequences recognized by high affinity anti-dsDNA Ab of SLE patients. SLE sera were used to bind templates from a pool of double-stranded oligonucleotides (ON). A central part of 20 base-pair random sequence was flanked by restriction endonuclease recognition sites and sequences complementary to predefined PCR primers. Immunoselected ON were precipitated, isolated from the immune complexes and then subjected to a further immunoprecipitation step after amplification by PCR. After five cycles of immunoprecipitation and PCR, the resulting ON were cloned. Sequence analysis revealed that sera from SLE patients and two human monoclonal anti-dsDNA Ab obtained from SLE patients preferentially select sequences expected to form non-B-DNA structures. Inhibition studies of the Farr assay confirmed the increased affinity of the selected epitopes for anti-DNA Ab as compared to random B-DNA.     

生物芯片技术与基础医学研究

典型的生化分析系统通常包括三个部分：样品制备、生化反应和结果检测。许多年来,如何使这三个部分成为有机的结合体一直是许多科学工作者和企业界人上的梦想。生物芯片——分子生物学和半导体工业的完美结合——使得这一梦想成为了现实。生物芯片是应用于生命科学和医学领域中作用类似于电子芯片的器件,是便携式生物化学分析器的核心技术。通过对微加工获得的微米结构做生物化学处理能使成千上万个与生命相关的信息集成在一块厘米见方的芯片上。生物芯片将生命科学中许多不连续的过程如样品制备、化学反应和结果检测步骤移植到芯片上并使…     

Single-cell electroporation using proton beam fabricated biochips

We report the design and fabrication of a novel single cell electroporation biochip featuring high aspect ratio nickel micro-electrodes with smooth side walls between which individual cells are attached. The biochip is fabricated using Proton Beam Writing (PBW), a new direct write lithographic technique capable of fabricating high quality high-aspect-ratio nano and microstructures. By applying electrical impulses across the biochip electrodes, SYTOX? Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells and observed via green fluorescence when the stain binds with DNA inside the cell nucleus. Three parameters; electric field strength, pulse duration, and numbers of pulses have been investigated for the single cell electroporation process. The results indicate high transfection rates as well as cell viability of 82.1 and 86.7% respectively. This single cell electroporation system may represent a promising method for the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.     

Genomic cloning and partial characterization of human chymotrypsinogen gene

Summary Chymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescencein situ hybridization and G-banding analysis.     

Screening for the breast cancer gene (BRCA1) using a biochip system and molecular beacon probes immobilized on solid surfaces

We describe the use of a biochip based on complementary metal oxide semiconductor (CMOS) technology for detection of specific genetic sequences using molecular beacons (MB) immobilized on solid surfaces as probes. The applicability of this miniature detection system for screening for the BRCA1 gene is evaluated using MB probes, designed especially for the BRCA1 gene. MB probes are immobilized on a zeta-probe membrane by biotin-streptavidin immobilization. Two immobilization strategies are investigated to obtain optimal assay sensitivity. The MB is immobilized by manual spotting on zeta-probe membrane surfaces with the use of a custom-made stamping system. The detection of the BRCA1 gene using an MB probe is successfully demonstrated and expands the use of the CMOS biochip for medical applications.     

Development of a TaqMan real-time RT-PCR assay for the detection of rabies virus

Diagnosis of rabies relies on the fluorescent antibody test (FAT) from brain impression smears. The mouse brain inoculation test is used to confirm FAT but requires weeks until the result is known. TaqMan real-time PCR has been described for rabies viral RNA detection; however, this is burdened by primer and probe binding site mismatches. The purpose of this study was to develop a TaqMan real-time RT-PCR assay as an adjunct to FAT, based on national data of 239 rabies nucleoprotein sequences from rabies-infected brain specimens collected between 1998 and 2003. Two showed as many as 3 mismatches. However, mismatches on primer and/or probe binding sites did not affect amplification or detection. One hundred and forty-three brain samples submitted for rabies diagnosis from all over the country between 2005 and 2007 were also tested. Results were concordant with FAT. Thirteen rabies proven samples from Myanmar, Cambodia, Indonesia and India; 3 of which had up to 7 mismatches at primer/probe binding sites, could be detectable. Challenge Virus Standard, a fixed virus strain with 4 mismatches at probe binding site, could not be detected but remained amplified. This assay could be used as an adjunct to FAT and may serve as a rabies surveillance tool.     

Identification of sequences in rotavirus mRNAs important for minus strand synthesis using antisense oligonucleotides.

The core of the rotavirion consists of three proteins, including the viral RNA polymerase, and 11 segments of double-stranded (ds)RNA. The RNA polymerase of disrupted (open) cores is able to catalyze the synthesis of dsRNA from exogenous viral mRNAs in vitro. In this study, we have identified sequences in exogenous viral mRNAs important for RNA replication using antisense oligonucleotides. The results showed that oligonucleotides complementary to the highly conserved 3'-terminal sequence of rotavirus mRNAs prevented all but basal levels of dsRNA synthesis. Notably, we observed that the addition of oligonucleotides which were complementary to nonconserved sequences present either at the 5'- or 3'-end of a viral mRNA effectively inhibited its replication without interfering with the replication of other viral mRNAs present in the same replication assay. Thus, the nonconserved sequences in rotavirus mRNAs contain gene-specific information that promotes RNA replication. The fact that antisense oligonucleotides inhibited dsRNA synthesis indicates that the strandedness (single- versus double-stranded) and secondary structure of the viral mRNA template are factors that affect the efficiency of minus strand synthesis.