东方粘虫(Pseudaletia separata(Walker))微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用包被链亲和素的磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)15、(GA)15、(GTT)12、(GAT)12、(TAGA)8 和 (GTGA)8退火结合的含接头和微卫星序列的单链粘虫基因组DNA限制性酶切片段,获得单链目的片段.经PCR扩增形成双链后连接到pGEM-T 载体上,再转化到DH5α热感受态细胞中,首次成功构建粘虫基因组微卫星富集文库.随机抽样(16次抽样,每库共12～114个克隆)测序发现,6个文库总的微卫星阳性克隆率30.07%,CA/GAT/GTT 等3个文库的阳性克隆率达到40%以上.单次抽样最高阳性克隆率达到56%.粘虫微卫星富集文库的建立和高多态性微卫星单拷贝位点的筛选将为粘虫的生态遗传学研究、连锁图谱构建、分子进化和系统发育研究等提供大量遗传标记,对昆虫迁飞的分子生态研究也有重大意义.     

两种书虱微卫星富集文库的构建及比较

利用链霉亲和素与生物素之间的强亲和性原理,将链霉亲和素偶联的磁珠与微卫星探针(AC)12、(TC)12、(ATC)8、(ATG)8、(AAC)8、(ATAC)6及(GATA)6退火结合后,再亲和捕捉含接头和微卫星序列的单链书虱基因组DNA限制性酶切目的片段,经PCR扩增形成双链后进行克隆、建库。结果表明本研究成功构建了嗜卷书虱和嗜虫书虱共13个微卫星富集文库,包括6个嗜卷书虱文库,7个嗜虫书虱文库,其平均阳性克隆率为71.17%。经检测发现共得到两种书虱260个微卫星位点。这两种书虱微卫星富集文库的建立和高多态性微卫星位点的筛选将为嗜卷书虱和嗜虫书虱的种群遗传与进化、基因连锁图谱构建、分子系统发育研究等提供大量分子遗传标记,对其在实仓中的持续控制提供遗传学信息。     

牦牛基因组微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用链亲和素磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)12、(CCG)8、(CAG)8、(TTTC)8退火结合的含有接头和牦牛微卫星序列的单链限制性酶切片段,获得单链目的片段,经PCR扩增形成双链,然后克隆到pMD18-T载体上,转化至DH5α中,首次成功构建牦牛基因组微卫星富集文库。测序结果发现,阳性克隆率为77％(37／48),说明构建的牦牛基因组微卫星富集文库是一个高质量的文库。牦牛富集微卫星文库的建立和牦牛微卫星的筛选将为下一步进行牦牛基因组结构的分析、牦牛遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。     

云南松基因组微卫星富集文库的构建

采用磁珠富集法构建云南松微卫星富集文库。云南松基因组DNA经RsaⅠ酶切,与特定接头连接,再用接头特异引物进行PCR扩增。连接扩增产物与用生物素标记的(AG)12、(AT)12、(CG)12、(GT)12、(ACG)12、(ACT)12和(CCA)8探针杂交,通过链霉亲和素偶联的磁珠捕捉含接头和微卫星序列的片段并扩增,将获得的片段连接到pMD-19T载体上,转化至大肠杆菌JM109感受态细胞中,成功构建了云南松微卫星富集文库。通过PCR检测从文库中筛选阳性克隆,在383富集阳性菌落中获得阳性克隆257个,经测序分析,在获得的159条序列中,有143条含有SSR,其中完美型占65.73%,非完美型占23.78%,混合型占10.49%。结果表明,磁珠富集法构建云南松基因组微卫星文库高效、可行的,文库的构建为微卫星位点的分离、遗传多样性的分析等奠定基础。     

兔(AG)n微卫星DNA富集文库的构建与鉴定

目的:微卫星遗传标记具有数量大、分布广且多态信息含量高等优点,因而被广泛用于动植物遗传图谱的构建、QTL定位、标记辅助选择及亲缘关系鉴定等领域。方法:采用亲和捕捉法。结果:构建了兔(AG)n微卫星DNA序列的富集文库,文库含重组克隆4 850个,其中含有(AG)n微卫星DNA序列的阳性克隆占66.7%。     

藏酋猴微卫星富集文库的构建及微卫星分子标记的筛选

通过磁珠富集法构建了藏酋猴AC重复和AAAG重复的微卫星富集文库,分离微卫星序列并对其进行分析。将藏酋猴基因组DNA经Sau3AI酶切后纯化回收,连接特定接头。用生物素标记的探针与酶切片段杂交,捕获300～1000bp片段,随后将获得的片段连接到pMD-19T载体上,转化至JM109中,成功构建藏酋猴微卫星富集文库。(AC)n富集文库和(AAAG)n富集文库的阳性克隆率分别为50%和10%左右。根据测序得到的48个微卫星序列成功设计了24对引物,最终筛选出6个微卫星标记,这些标记将为藏酋猴的遗传多样性研究、圈养种群结构的分析和遗传图谱的构建等奠定一定的基础。     

基于磁珠富集法的绣线菊SSR分子标记分离与筛选

微卫星序列（SSR）具有多态性高、共显性遗传等特点,是一种极具价值的分子遗传标记。采用磁珠富集法从高山绣线菊基因组DNA中分离和筛选SSR标记。高山绣线菊基因组经限制性内切酶Mse I酶切后与接头连接,并与生物素标记SSR探针（AC）15和（AG）15杂交,然后通过链霉亲和素磁珠富集、洗脱、PCR扩增、克隆,完成微卫星文库构建。利用载体通用引物和探针序列引物进行PCR扩增,筛选重组克隆并测序,获得112条序列。随机挑选其中60条序列设计的引物,经初期筛选获得多态性引物16对。用所得16对引物对4个居群92个个体的蒙古绣线菊和高山绣线菊进行PCR扩增。统计分析PCR产物的毛细管电泳结果,发现4个居群的平均等位基因数、平均期望杂合度及平均观测杂合度都比较高。64个数据系列（4个居群×16个位点）中的26个显著偏离HardyWeinberg平衡,推测可能由于无效等位基因的存在所引起。分析显示研究开发的16对多态性SSR引物可以用于后续遗传多样性、物种进化与亲缘关系等方面研究,丰富了绣线菊遗传多样性研究的分子标记。     

细梢小卷蛾微卫星富集文库的构建与分析

目的:构建细梢小卷蛾Rhyacionia leptotubula微卫星富集文库.方法:提取细梢小卷蛾基因组DNA,经限制性内切酶Rsa Ⅰ酶切,用(CT)10和(GT)10生物素探针与其杂交,利用磁珠富集含有微卫星的DNA序列,并对其进行PCR扩增,将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星富集文库:结果:对100个克隆进行随机测序,获得98个微卫星序列,其中具有5次及以上碱基重复次数的微卫星克隆占26%,最高碱基重复次数为33次,非完美型占12%,说明构建的细梢小卷蛾微卫星富集文库是一个高质量的文库.结论:该文库的建立为后续筛选具高多态性的微卫星标记引物研究细梢小卷蛾的种群遗传结构、迁移扩散规律等奠定了基础.     

鲫鱼微卫星DNA的分离及多态性分析

目的：用近缘物种鲤微卫星引物来分离鲫鱼微卫星标记并对其多态性进行分析。方法：以鲫鱼基因组DNA为模板,采用6对鲤微卫星引物进行PCR扩增,PCR产物经8%的非变性聚丙烯酰胺凝胶电泳和银染色检测。结果：筛选出2个以AC和TA为重复单元的鲫鱼新的微卫星标记。多态性分析表明,这2个微卫星标记的遗传杂合度分别为0.611和0.644,多态信息含量为0.536和0.572,属于高度多态性标记。结论：该研究筛选的2个微卫星标记可应用于鲫鱼遗传多样性、遗传连锁图谱构建及分子标记辅助育种等方面的研究。     

东方田鼠微卫星标记的富集筛选与初步应用

根据生物素与链霉亲和素的亲和原理, 利用磁珠富集法筛选东方田鼠(Microtus fortis)微卫星分子标记。链霉亲和素磁珠捕获生物素标记的微卫星探针, 然后与连有接头的单链限制性酶切片段复性结合, 获得含有微卫星的单链片段, PCR扩增形成双链, 连接T载体并转化感受态细胞, 得到东方田鼠微卫星富集文库。随机挑选70个阳性克隆, 经测序分析, 获得微卫星序列92个。设计合成27对微卫星引物并成功筛选出21对可用引物, 取其中10对引物, 荧光标记后对3个人工驯养及野生东方田鼠种群进行遗传多样性分析。结果显示, 文章所构建的东方田鼠微卫星文库的阳性克隆率较高, 初步筛选的10个微卫星标记均为具有高度多态性的微卫星标记。在3个东方田鼠种群中, 野生湖南种群的观测等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)和多态信息含量(PIC)均最高, 人工驯养的湖南种群次之, 人工驯养的宁夏种群最低。     

Sorting of chromosomes by magnetic separation

Summary Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy.     

Specific detection of residual CHO host cell DNA by real-time PCR.

Chinese hamster ovary cells have been widely used to manufacture recombinant proteins for human therapeutic use. A sensitive quantitative real-time polymerase chain reaction assay for the detection of residual Chinese hamster (Cricetulus griseus) DNA is presented in this paper. The assay is reasonably affordable and can be adapted for high-throughput screening using 96-well format. Real-time PCR primers were designed to amplify a 150bp region of a genomic fragment from hamster DNA. The specificity of the probe was evaluated in real-time PCR reactions using genomic DNA from mouse fibroblast, human kidney and hamster ovary cell lines as template. Sensitivity of real-time PCR was compared on genomic DNA from hamster cell line CHO DG44. These primers can be used in real-time PCR reactions to detect presence of contaminating hamster DNA in purified protein samples down to sensitivity of 300fg genomic DNA.     

A method for nucleic acid hybridization to isolated chromosomes in suspension

Summary A procedure was developed to provide differential fluorescent staining of metaphase chromosomes in suspension following nucleic acid hybridization. For this purpose metaphase chromosomes were isolated from a Chinese hamster x human hybrid cell line. After hybridization with biotinylated human genomic DNA, the human chromosomes were visualized by indirect immunofluorescence using antibodies against biotin and fluoresceine-isothiocyanate-(FITC)-labeled second antibodies. This resulted in green fluorescent human chromosomes. In contrast, Chinese hamster chromosomes revealed red fluorescent staining only when counterstained with propidium iodide. Notably, interspecies chromosomal rearrangements could be easily detected. After hybridization and fluorescent staining, chromosomes still showed a well-preserved morphology under the light microscope. We suggest that this procedure may have a useful application in flow cytometry and sorting.     

用Touch-Up PCR克隆中国仓鼠存活蛋白基因及其序列分析

目的:通过Touch-UpPCR(上升PCR)的方法从中国仓鼠卵巢细胞中克隆存活蛋白基因,并对其进行序列分析。方法:分别从293、H22、CHO-S细胞中提取总RNA并反转录得cDNA,然后用Touch-UpPCR克隆存活蛋白基因。结果:获得了存活蛋白基因,全长423bp,序列测定及同源性分析表明其与GenBank中报道的人以及小鼠的存活蛋白基因高度同源,同源性分别为84.8%和88.2%;氨基酸序列分析表明它们的蛋白产物具有功能相同的BIR结构域。结论:从中国仓鼠卵巢细胞中首次克隆到存活蛋白基因。     

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-phenylindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

Summary The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-pheny-lindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.     

Evolutionary relationships of the Chinese hamster X chromosome and autosomes: a comparison using solution hybridization techniques

The evolutionary relationships of Chinese hamster X chromosome and autosome DNA sequences were compared by solution hybridization techniques. Chinese hamster X chromosome tracer was prepared by radiolabeling DNA from chromosomes isolated by fluorescence-activated sorting. Radiolabeled Chinese hamster total genomic DNA, approximately 90% of which is of autosome origin, was used as autosome tracer. Each tracer was mixed with excess driver DNA of Chinese hamster, Syrian hamster, rat, rabbit, cat, cow, or human origin. Reaction mixtures were melted and allowed to reassociate to an equivalent CoT of 12000, under conditions which permitted 35% mismatch in DNA duplexes. Both the extent of duplex formation (the normalized percentage hybridization or NPH) and the average thermal stability of the duplexes formed (melting temperature or Tm) were measured; these values were used to compare the evolutionary relatedness of tracer and driver DNAs. The pattern of evolutionary relatedness revealed by comparing either the Tm or NPH values obtained with different drivers was the same for X chromosome and autosome DNA and was consistent with the phylogeny of the species examined. Although NPH and Tm values for X chromosome and autosome tracers differed, differences fell within the range of experimental error. The results of these studies provide no evidence for differential conservation of Chinese hamster X chromosome sequences, suggesting that the constraints on the mammalian X chromosome which act to maintain its gene linkage group intact do not markedly reduce the extent to which its sequences diverge during evolution.