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。     

黑麦1R染色体微克隆文库的构建与分析

通过玻璃针分离法 ,从黑麦 (SecalecerealeL .)根尖细胞中期分裂相中显微分离出 2条及 5条 1R染色体。经Sau3A接头介导的PCR(LA_PCR)方法对其进行体外扩增 ,得到了 0 .3～ 2 .5kb之间的DNA片段。以DIG标记的探针进行多次Southern杂交 ,证明显微分离出的染色体的体外扩增产物与黑麦基因组DNA同源 ,并且来自 1R染色体。然后利用 5条 1R染色体的第二轮PCR产物构建质粒文库 ,可得到 2 2 0 0 0 0个重组子。随机挑选 172个重组子进行分析 ,发现插入片段主要介于 30 0～ 180 0bp之间。此外 ,根据基因组点杂交结果推算出该文库包含约 42 %的中、高重复序列和 5 8%的单、低拷贝序列 ,而且文库的冗余度较低。研究构建的黑麦 1R染色体微克隆文库为 1R染色体高密度遗传图谱的建立以及位于其上的重要基因的定位与分离提供了便利。     

小麦6B染色体微切割及其不同片段的DNA文库构建

用Nd:YAG激光微束将处于丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收.将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增.Southem杂交证明4个特定区域的DNA确实来自于小麦基因组.用一系列(42对引物)位于6B染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证.结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体.将6B染色体4个片段的第二轮PCR产物克隆到pGET-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×105、2.74×105和2.93×105个重组子克隆.每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证.结果显示;插入片段大小在300～1800之间,平均大小为820～870bp,其中43%～48%的克隆为低/单拷贝序列,42%～47%为中/高拷贝序列.本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础.     

小麦6B染色体微切割及其不同片段的DNA文库构建

用Nd∶YAG激光微束将处于有丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收。将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增。Southern杂交证明4个特定区域的DNA确实来自于小麦基因组。用一系列(42对引物)位于6B染色体和其他染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证。结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体。将6B染色体4个片段的第二轮PCR产物克隆到pGEMT-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×10~5、2.74×10~5、2.45×10~5和2.93×10~5个重组子克隆。每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证。结果显示:插入片段大小在300～1800 bp之间,平均大小为820～870 bp,其中43％～48％的克隆为低/单拷贝序列,42％～47％为中/高拷贝序列。本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础。     

应用RD-PCR技术制备HIV基因芯片探针

利用限制性显示 (RD PCR)技术快速分离HIV 1基因片段制备DNA芯片探针 .以Sau3AⅠ酶切HIV基因 ,得到许多大小适合芯片的限制性酶切片段 .然后在片段两端接上接头 ,根据酶切位点、接头的序列设计通用引物 .在该通用引物的 3′端分别延伸一个碱基后 ,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 .阳性克隆经鉴定、扩大培养后提取质粒 .以质粒为模板扩增靶片段并进行序列分析 .每个亚型得到了十几个 10 0～ 10 0 0bp的HIV基因片段 .研究表明 ,RD PCR技术是一种有效的快速制备基因芯片探针的方法     

水仙单染色体特异文库的构建

采用玻璃针分离法,通过显微操作系统成功地分离到一个多花水仙(N arcissus tazetta L.,2n=22)品种的中着丝粒单染色体,将分离到的单染色体放入0.2 mL Eppendorf管中,经去蛋白、S au3A酶切,并在染色体DNA片段两端加上S au3A人工接头后,进行两轮PCR扩增,得到0.3~3.0 kb之间的DNA片段.用水仙基因组DNA标记作探针,与扩增产物进行Sou thern杂交,从而证明单染色体DNA确实已被成功地扩增.将第二轮PCR产物构建质粒文库,随机挑取90个重组子进行分析,发现插入片段主要在600~2 500 bp之间.     

背角无齿蚌基因组(GT)n微卫星DNA特征

利用磁珠富集法筛选背角无齿蚌(Anodonta woodiana)的微卫星分子标记,采用Sau3A1酶对完整DNA进行酶切,以生物素标记的(GT)15寡核苷酸探针从酶切片段中筛选微卫星序列.洗脱的杂交片段克隆到PGEM-T载体上构建富集微卫星基因组文库后,通过菌液PCR筛选检测出阳性克隆进行测序.结果表明:在筛选的18...     

纵纹腹小鸮DNA指纹谱探针的筛选和初步应用

以人工合成的微卫星序列 (GTG) 5,(GT) 8,(CAC) 5和人源小卫星 33 1 5作引物 ,扩增纵纹腹小的基因组DNA ,产生多态性DNA片段 ,回收了 8个表现个体特异性的片段。当用小的基因组总DNA探针与它们杂交时 ,其中 2个表现阳性 ,说明PCR方法扩增出的高变异产物含有重复序列。用含重复序列的个体特异性PCR产物作探针 ,与无关个体小基因组DNA的HaeⅢ酶切产物进行DNA印迹 ,获得了变异性较高的DNA指纹图谱。且通过对京白鸡家系分析表明 ,用小基因组DNA的PCR产物分离制备的探针所获得的DNA指纹图带能够稳定的遗传。因此 ,高变异的PCR产物可以有效地用作DNA指纹探针。     

炭疽芽胞杆菌基因芯片探针文库的构建

为制备炭疽芽胞杆菌的基因芯片探针文库,以炭疽芽胞杆菌毒素质粒pX01和荚膜质粒pX02为原材料,用Sau3A I酶切pX01和pX02质粒DNA,Taq DNA聚合酶72℃补平加A,经AT克隆,PCR初步鉴定筛选出炭疽质粒片段的阳性克隆．DNA自动分析仪对克隆片段进行序列测定;用生物信息学方法对其片段进行同源性分析;并将克隆的探针打印于玻片上,制备成炭疽芽胞杆茵基因芯片,与炭疽杆茵质粒DNA样品进行初步芯片杂交的实验,杂交实验的阳性率达到了90％以上,证明大部分克隆探针属于炭疽芽胞杆菌．炭疽芽胞杆菌基因芯片探针文库的构建为基因芯片探针的制备摸索出一条简便、高效、可行的方法．     

不吸水链霉菌基因文库的构建及初步筛选

采用碱裂解法从不吸水链霉菌提取染色体DNA,用Sau3AI对染色体DNA进行部分水解,利用透析袋法回收3.5～9kb之间的DNA片段。以pUC18为载体,用BamHI对其进行酶切,再用热敏磷酸酶去磷酸化,酶切产物与外源DNA按一定比例混合后,加入T4DNA连接酶进行连接。连接产物用受体菌E.coliDH5α进行转化,根据α-互补性质产生的颜色反应,挑选白色菌落,构建了相应的不吸水链霉菌基因文库。分别利用特异性探针2-1-1及1-2-1对不吸水链霉菌基因文库进行筛选,利用DIG-Ⅱ-dUTP对特异性探针进行标记,与基因文库中的阳性转化子进行Southern杂交,通过显色反应对杂交结果进行检测,由于时间关系,暂未筛选出阳性克隆,但这些工作对以后的相关实验研究具有很好的借鉴作用。     

斯卑尔脱小麦1B染色体的显微分离及其DNA的PCR扩增

以斯卑尔脱小麦的一个变种及其近等基因系为材料,玻璃针法进行显微操作在减数分裂中期I分裂相中获得G型细胞质雄性不育的育性恢复基因Rf₃所在的1B染色体DNA。LA-PCR方法扩增DNA,斑点杂交的方法检测证明扩增产物来源于斯卑尔脱小麦基因组的1B染色体DNA。     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Two and five 1R chromosomes were microdissected from the metaphase spreads of rye ( Secale cereale L. ) root-rip cells with the aids of glass needles. The dissected chromosomes were amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 0.3 to 2.5 kb smear DNA fragments were obtained. After hybridized with DIG labeled probes, it was confirmed that the PCR products of the microdissected chromosomes were homologous with the rye genomic DNA, and derived from the 1R chromosome as well. Then, the second round PCR products from five chromosomes of 1R were microcloned to construct the plasmid library, including 220 000 clones. 172 randomly selected clones were evaluated ranged in size from 300 to 1 800 bp. Furthermore, the genomic dot hybridization results indicated that the library contained nearly 42% medium/high repetitive sequences and 58% low/single copy sequences, and its redundancy was very low. In this research, many aspects of the 1R chromosome microclone library exceeded or approached those of the previous reports in the literatures. Those are potential for construction of a high density genetic map of chromosome IR, from which some important genes can be tagged and isolated.     

Microdissection and Microcloning of Chromosome 5 in Gossypium arboreum

Construction of single chromosomal DNA libraries by chromosome micromanipulation is a useful tool for pursuing genomic studies. Thus far, micromanipulation in cotton has not been reported yet, which may be due to difficulty in preparing chromosomes of similar sizes. In this study, single chromosome micromanipulation was successfully achieved in cotton. A single chromosome 5 of Gossypium arboreum (cultivar Shixiya-1) carrying a large satellite at mitotic metaphase was isolated by microdissection using the Cell Cut Plus Laser micromanipulation system. The chromosomal DNA was digested by Sau 3A and ligated to Sau 3A linker adaptors. After two rounds of linker adaptor PCR (LA-PCR) amplification, DNA fragments ranging from 300 to 2,500?bp were acquired. Southern hybridization revealed that the PCR products had homology with genomic DNA of the cultivar Shixiya-1, indicating that DNA of chromosome 5 has been successfully amplified. The second round LA-PCR products and 45S rDNA and chromosome-specific BAC clones were used as probes for fluorescence in situ hybridization analysis on metaphase chromosome. The results confirmed that the LA-PCR products were derived from the isolated target chromosome. Hybridization signals of the second round LA-PCR products were mainly detected along the entire chromosome 5; in addition, weak signals were also observed on other chromosomes, indicating that there were some homologous nucleotide sequences in other chromosomes. The second round LA-PCR products were cloned to generate a chromosome-specific DNA library which contains approximately 173,000 clones. Evaluation based on 136 randomly selected clones showed that the size of the inserts varied from 500 to 1,800?bp with an average of 750?bp. The no-load rate was less than 1?%, the titer of the library was 1.2?×?10⁶ pfu mL^?1, and the rate of the single and low copy sequences was over 47?%. This library will facilitate specific probe screening, molecular mapping, gene cloning, and DNA sequencing for this chromosome.     

Development of microdissection and chromosome specific genomic library in Lilium tigrinum

Techniques for microdissection and microcloning were established using chromosome 1 of triploid Lilium tigrinum. Chromosome 1 was dissected from a membrane slide using a microbeam system. Digestion with proteinase K was done before PCR amplification for more than 24 hours. The dissected chromosomes were then amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and linker adaptor-mediated PCR (LA-PCR). Successful PCR amplification relied on critical concentrations of both MgCl₂ and Taq polymerase. The optimum concentration of MgCl₂ and Taq polymerase was 2.5 mM and 0.5 U, respectively. Amplification of the dissected chromosome using DOP-PCR had more sensitivity dependent upon PCR factors, but LA-PCR was more dependent on the linker ligation. Amplified DNA products ranged from 100 to 2500 bp both for DOP- and LA-PCR. Evaluated clones only ranged from 100 to 1700 bp for DOP-PCR and 100 to 900 bp for LA-PCR. Based on the sequence results, most of the sequences from the DOP-PCR and LA-PCR showed no significant similarity with known data in NCBI database. However, about 2% of the sequence data was partially matched with plant microsatellites with low similarity. The results derived from the microdissection of a large genome organism such as Lilium showed informative and useful for the development of microsatellite repeat markers. Sequence data from the chromosome specific DNA library was considered for the development of microsatellite markers.     

Generation of PCR-based markers for the detection of rye chromatin in a wheat background

Oligonucleotide primers were developed to detect the presence of four rye sequences using a PCR assay. These assays give a rye-specific signal from wheat DNA template which contains various rye chromosomes or chromosome segments. The sequences identified were associated with the nucleolar organiser region, the 5S-Rrna-R1 locus, the telomere, and a widely dispersed, rye-specific repetitive element Ris-1. The primers amplified from the well-established loci Nor-R1 and 5S-Rrna-R1 on rye chromosome arm 1RS, and also located a 5s-Rrna locus on chromosome 3R. The telomere-associated sequence was present on every rye chromosome, and was also present, at a low copy number, in both wheat and barley. These assays will be particularly useful for introgression programmes aimed at reducing the rye content of the 1BL.1RS wheat-rye translocation. When multiplexed, the primers will enable a rapid, simultaneous assay for a number of distinct rye loci, which can be derived from a small portion of mature endosperm tissue.     

Microdissection and PCR Amplification of Single Soybean Chromosome

Glass needles were successfully used to dissect the soybean (Glycine max L. ) single chromosome under the micromanipulator in this research. Two dissected soybean chromosomes were digested by Sau3A in two 0.5 mL Eppendorf tubes respectively. The two ends of chromosomal fragments were ligated with Sau3A linker adoptor. After two rounds of PCR amplification, smear DNA fragments ranged from 0.3 to 3 kb were acquired. Southern hybridization result showed the PCR products from the two single soybean chromosomes were homogeneous with the soybean genomic DNA, indicating that DNAs from the two single chromosomes have been successfully amplified. At the same time, the amplified products from the two of the distinguished single chromosome appeared somewhat different. The authors dissected the small chromosomes only by a traditional inverted microscope. Therefore, this research provides a plausible chance for amplification and microcloning of single small chromosomes.     

Sau3A in situ digestion of human chromosome 3 pericentrometric heterochromatin. I. Differential digestion of alpha-satellite and satellite 1 DNA sequences.

In situ digestion with the restriction endonuclease (RE) Sau3A (Sau3A REISD) uncovers a polymorphism for the pericentromeric heterochromatin of human chromosome 3, which can be positively stained (3+) or not (3-), and has proven useful to differentiate donor and recipient cells after sex-matched bone marrow transplantation and to analyze the so-called hemopoietic chimerism. The aim of the present investigation was to obtain insight into the molecular basis of such polymorphism to optimize its use for chimerism quantification using methodological approaches other than REISD. To this end, fluorescence in situ hybridization (FISH) assays using probes for the satellite DNA sequences that mainly constitute chromosome 3 pericentromeric heterochromatin (alpha-satellite and satellite 1 DNA) were performed on control and Sau3A-digested chromosomes. The results obtained suggest that chromosome 3 alpha-satellite DNA is digested in all individuals studied, irrespective of the karyotype obtained by Sau3A REISD (3++, 3+-, 3--), and thus it does not seem to be involved in the polymorphism uncovered by Sau3A on this chromosome. Satellite 1 DNA is not digested in any case, and shows a polymorphism for its domain size, which correlates with the polymorphism uncovered by Sau3A in such a way that 3+ chromosomes show a large domain (3L) and 3- chromosomes show a small domain (3S). It seems, therefore, that the cause of the polymorphism uncovered by Sau3A on the pericentromeric region of chromosome 3 is a difference in the size of the satellite 1 DNA domain. Small satellite 1 DNA domains fall under the resolution level of REISD technique and are identified as 3-.