从水稻细胞核中制备分子量达到Mb级的DNA

用水稻黄化苗作实验材料,经蔗糖不连续梯度纯化细胞核、琼脂糖包埋、蛋白酶消化释放DNA,脉冲交变电泳显示所得DNA样品分子量在200kb至3Mb之间,其中大量集中在2.2Mb处.所得DNA容易被多种内切酶消化和连接,可用于构建水稻YAC文库和大尺度物理图的研究.     

玉米单染色体的分离和体外扩增

建立了玉米单染色体的分离及体外扩增的方法。取95％乙醇固定后经果胶酶和纤维酶酶解的根尖制备染色体标本,用自制的微细玻璃针在倒置显微镜下挑取目的染色体。染色体DNA经Sau3A酶切后与人工合成的Sau3A连接接头连接,经两次PCR扩增获得足以用于构建单染色体DNA文库的扩增产物。片段大小为0．3～5kb,多数为0．5～3．5kb．与前人研究方法相比,所需底物量少（只需1条染色体）,扩增片段大,为植物中小型染色体分离、体外扩增进而进行单染色体DNA文库构建奠定了基础。     

温敏核不育水稻5460S细菌人工染色体文库的构建和鉴定

为了构建温敏核不育 (TGMS)基因区域的精细物理图谱并最终分离TGMS基因 ,以温敏核不育水稻 5 46 0S为材料 ,摸索优化了构建植物细菌人工染色体 (BAC)文库的方法 ,构建了一个高质量的BAC文库 .该文库由 1 95 84个克隆构成 ,插入片段平均长度为 1 1 0kb ,相当于水稻单倍体基因组大小的 5倍 ;以分子量分别为 1 40和2 5 0kb的 2个大BAC克隆进行稳定性传代实验 ,经 1 0 0代传代后其插入的DNA片段仍然稳定存在 ;以线粒体和叶绿体基因为探针筛选BAC文库 ,未检验出叶绿体和线粒体DNA的污染 ;以和tms1基因连锁的 3个分子标记作为探针对BAC文库进行了筛选 ,每个探针至少可获得一个阳性克隆 ,利用热不对称性交错PCR(Tail PCR)法成功分离了阳性克隆的左右末端序列 .     

紫茎泽兰高分子量核DNA制备方法的研究

以紫茎泽兰暗培养幼嫩叶片为材料,在细胞核提取缓冲液中加入PVP、抗坏血酸和氨基甲酸钠3种强抗氧化剂,较好地去除了多糖多酚等次生代谢物,经低熔点琼脂糖包埋细胞核及蛋白酶K原位裂解DNA胶块,以获得大分子量DNA;最后分别采用2种不同的限制性内切酶(BamH Ⅰ和HindⅢ)和5个浓度梯度(0 U、0.2U、0.4U、0.6U、0.8 U)进行酶切,并对不同的回收方法进行比较分析.结果表明:经脉冲电泳检测,本方法提取的DNA片段长度＞600 kb,基本无细胞器DNA和蛋白质污染;酶切条件以0.4U酶浓度HindⅢ的效果最好,酶切后的DNA片段主要集中在100～300 kb之间,DNA片段大小满足构建BAC文库的要求;对回收方法进行比较分析,电洗脱法回收的大片段DNA浓度远远大于琼脂糖酶切法,大于8mg·L-1,这完全能够满足构建大片段基因组文库对DNA浓度的要求.     

小麦条锈菌大片段基因组DNA的提取方法研究

由条锈菌Puccinia striiformis引致的小麦条锈病是小麦最重要的病害之一。由于其活体寄生的特点,对小麦条锈菌的遗传学和分子生物学研究十分有限,大片段核DNA的提取研究还未见报道。高分子量基因组DNA是开展大片段基因组文库构建、基因组分析以及基因组重建的重要基础,通过系统建立和优化小麦条锈菌大片段基因组DNA的分离方法,成功获得分子量大于1Mb高质量的病菌基因组DNA。     

细菌人工染色体文库的构建方法分析

细菌人工染色体是一种承载大片段DNA的新型载体系统,它具有插入片断大、嵌合率低、遗传稳定性好、易于操作等优点.在高等生物基因组文库的构建和基因功能的分析等方面有广泛应用.BAC文库的构建是基因组较大的真核生物基因组学研究的重要基础.介绍了近年来细菌人工染色体文库构建方法上的研究进展,对其中载体的制备和高分子量DNA的制备这两个关键环节作了较深入的探讨.     

水稻中期染色体和DNA纤维的高效制备技术

水稻中期染色体和DNA纤维制备是水稻分子细胞遗传学研究中的关键技术。目前,这两个技术还有很多不足,该研究建立了高效制备水稻中期染色体和DNA纤维的方法。该方法制备的染色体,分裂相多、杂质少、背景清晰、染色体分散且形态好,水稻根尖分生组织细胞的分裂指数高达25％。植物细胞的细胞壁是制备DNA纤维的最大障碍,所以必须先提取细胞核,然后裂解细胞核释放出DNA纤维。在这个研究中,还建立了一个用刀切法分离细胞核,进而用SDS裂解核膜,用载玻片拖出DNA来制备水稻DNA纤维的方法。该方法制备的DNA纤维多呈平行的细线,背景清晰,伸展的程度均匀,适合于原位杂交。     

洋葱伯克霍尔德氏菌HMW DNA的制备及酶切研究

高质量粘粒基因组文库构建的关键是HMW DNA的长度至少为粘粒载体容量的10倍,通常粘粒载体的容量为30～50 kb,因此提取的HMW DNA应不小于500 kb.HMW DNA在制备时不能受到任何物理的剪切力,以免DNA断裂和损伤.利用琼脂糖凝胶包埋制备的DNA胶块经裂解和纯化后发现其DNA长度远大于500 kb,明显优于商品化试剂盒提取和酚抽提法.用BamHⅠ、Sau3AⅠ、XbaⅠ和HindⅢ对DNA胶块进行不完全酶切研究表明,构建文库常用的Sau3AⅠ并不适合胶块内酶切反应,而BamHⅠ酶切B.cepacia HMW-DNA效果较好,产生的DNA片段集中在20～50 kb之间,完全适合粘粒基因组文库的构建,为B.cepacia大插入片段基因组文库的构建以及功能基因组的研究奠定了良好的理论基础.     

分子生物学技术讲座（四）——真核基因组DNA的制备,分析及基因组DNA文库的构建

真核细胞DNA的制备通常是在有EDTA及SDS一类去污剂的存在下用蛋白酶K消化细胞后用酚抽提而实现的。用此方法得到的DNA,其大小为IOO～15Okb,恰好用于SOIJthern分析和经限制性内切酶部分消化后用人噬体载体构建基因组DNA文库。而用粘粒载体或用酵母人工染色体（YAC）载体构建基因组DNA文库则需要更大的DNA。1关于DNA的制备制备真核细胞基因组DNA的方法有许多种,但基本步骤相类似,即包括组织匀浆、细胞裂解、蛋白和RNA的去除以及用乙醇沉淀或透析等方法除去残存的其它物质。方法的选择主要根据样品的类型以及所制备的DN…     

通过微切一扩增建立植物(蚕豆Vicia faba)染色体区段特异性基因文库研究初探

以蚕豆（Viciafaba,2n=12）根尖为材料,采用改良方法制备染色体标本,在光镜下切割分离一段大M染色体核仁组织区（NOR）特定区段（约合0．9pgDNA）,通过单一引物一聚合酶链式反应法（SingleUniquePrimer-PCR）随机扩增微切DNA后,获得近60μgDNA。经琼脂糖电泳分析测定扩增产物分子片段大小介于200-900bp。以地高新（Digoxigenin）标记蚕豆总体DNA,作为探针与扩增产物进行Southern杂交,证实扩增得到的DNA与蚕豆DNA同源,来自做切染色体。部分扩增产物经ECORI酶切后,连人经同酶切割后的pUC18载体质粒,转化大肠杆菌（EcoliJM109）。琼脂糖电泳分析得到的部分克隆,得知插人子长度介于0．25-0．9kb。本文将用于动物材料的单一引物一聚合酶链式反应法应用于植物染色体的微切微扩增,并作了一定程度简化,初步建立起一套包括微切、扩增、检测和克隆的便捷、经济的实验室制备植物染色体区域特异性基因文库的方法。     

Preparation of megabase-size DNA from plant nuclei

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low-melting-point agarose microbeads or plugs, and DNA purification in situ . This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed-field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.     

Preparation of HMW DNA from Plant Nuclei and Chromosomes Isolated from Root Tips

Simple, fast and cost-effective method for preparation of DNA with high molecular weight (HMW DNA) from plant nuclei and mitotic chromosomes has been developed. The technique involves mechanical homogenization of formaldehyde-fixed root tips, purification of nuclei and/or chromosomes on sucrose gradient, embedding in low-melting-point agarose, and DNA isolation in agarose plugs. Alternatively, nuclei and chromosomes may be purified using flow cytometry. Majority of DNA obtained is megabase-sized and well digestible by restriction endonucleases. The method is highly efficient as microgram amounts of DNA can be obtained from only several milligrams of plant tissue. Handling negligible amounts of plant material reduces the consumption of chemicals. Furthermore, the use of root tips makes it possible to obtain high-quality DNA even from plant species with leaves that are rigid or rich in secondary metabolites such as polyphenols. It is expected that preparation of HMW DNA from root tip nuclei will facilitate long-range mapping and construction of large-insert DNA libraries also in these species. Successful isolation of HMW DNA from flow-sorted chromosomes opens a way for construction of chromosome-specific large-insert libraries in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

A simple method for isolation of megabase DNA from cotton

A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps, (i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7 Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning.     

Isolation of megabase-size DNA from sorghum and applications for physical mapping and bacterial and yeast artificial chromosome library construction

A method was developed for the isolation of megabase-size DNA fromSorghum bicolor. Sorghum protoplasts were isolated from young leaf tissue, embedded in an agarose matrix as microbeads or plugs, followed by cell lysis and protein degradation. The DNA prepared by this method was larger than 1 Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries.     

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation

Quantitation of UV-induced DNA damages in nanogram quantities of non-radiactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.     

Construction and screening of BAC libraries made from Brachypodium genomic DNA

Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.     

Shotgun法测序制备高纯度水稻基因组DNA的方法探讨(英)

在用散弹 (shotgun)法测定水稻 (OryzasativaL .ssp .indica)基因组全序列的过程中 ,叶绿体和线粒体DNA的污染问题非常严峻 .应用脉冲场电泳 (PFGE)技术对水稻基因组DNA进行纯化 ,结果表明它能够有效去除叶绿体和线粒体DNA ,使其污染率从 3%降低到 0 2 % .同时 ,比较了水稻绿苗和黄化苗的DNA得率 ,以及HB法和NIB法制备大分子质量(HMW)DNA的异同 .最后提出一套制备水稻基因组DNA的方法 ,包括黄化苗培养 ;细胞核的分离、包埋和裂解 ;脉冲场电泳纯化、回收聚集在低熔点 (LMP)胶中的水稻HMWDNA .用该方法所得的水稻基因组DNA ,纯度高 (无叶绿体和线粒体DNA污染 )、基因组完整 (机械剪切和降解少 )、回收率高 (操作过程中DNA损失少 ) .另外 ,首次报道在融化的低熔点(LMP)胶中对水稻HMWDNA于 38℃进行超声波处理 ,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1 5～ 3kb ,3～ 12kb) ,并且效果优于在TE中进行操作     

Construction and characterization of a bacterial artificial chromosome library of peach

A peach [Prunus persica (L.) Batch] bacterial artificial chromosome (BAC) library of var. Jingyu was constructed. Jingyu is a traditional variety, that displays many of the important agronomic characters of stone fruits. Since peach leaves are rich in polysaccharides, high-molecular-weight (HMW) DNA was extracted from leaf nuclei using a protocol adapted to peach. The HMW DNA embedded in agarose plugs was partially digested by HindIII. After size-selection by pulsed field gel electrophoresis, the selected DNA fragments were ligated to pBeloBAC11 and transformed into E. coli DH10B cells by electroporation. In total 20,736 recombinant clones were obtained. The BAC library has an average insert size of 95 kb and represents approximately 6.7 peach haploid genome equivalents. The BAC clones were stable in E. coli cell after 100 generations. The lack of hybridization to chloroplast and mitochondrial genes demonstrated that the library is predominantly composed of nuclear DNA. The library was screened with two molecular markers, W4 and P20, that are linked to white flesh and nectarine genes of peach, respectively. Ten positive clones were detected. Their fingerprints will be used to determine clone relationships and assemble contigs. This library should be well-suited for the map-based cloning of peach genes and genome physical mapping. Received: 18 January 2000 / Accepted: 29 May 2000     

Isolation of HMW DNA from sunflower (Helianthus annuus L.) for BAC cloning

The cultivated sunflower (Helianthus annuus L.) is one of the most important oil crops in the world. The importance of sunflower oil in human nutrition and in the chemical industry makes the sunflower a major research interest. An essential element for genomic libraries is the preparation of high molecular weight (HMW) DNA. We developed 2 methods for isolating HMW sunflower DNA. We prepared the DNA from nuclei and from protoplasts isolated from mesophyll tissue with the enzymes cellulase RS and pectolyase Y23. The HMW DNA was digested with restriction endonucleases. The ethidium bromide-stained gel suggested the DNA to be completely digested. These results were confirmed by Southern analysis using a radiolabeled RFLP marker. Both methods made it possible to generate sufficient quantities of megabase-size sunflower DNA suitable for bacterial artificial chromosome (BAC) cloning.