曼氏无针乌贼GT微卫星位点的筛选

采用链霉亲和素包被的磁珠富集法筛选曼氏无针乌贼(Sepiella maindroni)微卫星位点。试验样品来自舟山六横岛,提取4个样品的DNA混合成DNA pool,用限制性内切酶Sau 3A I酶切。接上接头后构建基因组PCR文库,用生物素标记的(GT)15探针筛选。将筛选获得目的片段进行PCR扩增,连接pMD18-T载体,转入DH5α感受态大肠杆菌里,扩大培养后PCR筛选阳性克隆。总共选取278个克隆,对120个经过检测含有插入片段的克隆进行测序,发现102个克隆含有微卫星序列,阳性克隆比率为85%。除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有64条。     

Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.

In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

CUT&Tag产物回收和建库方法的优化

新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质免疫共沉淀测序(chromatin immunoprecipitation sequencing,ChIP-seq)的实验过程。CUT&Tag中转座酶完成标签化后需要DNA回收或其他后处理才能进行建库PCR,不同的回收方法对CUT&Tag结果有着显著的影响。通过建立生物素化转座体-链霉亲和素磁珠体系(streptavidin beads recovery CUT&Tag,srCUT&Tag),可以快速便捷地完成CUT&Tag的产物回收。本文在K562细胞中展开H3K4me3、RNA聚合酶Ⅱ(RNA polymeraseⅡ,RNAPⅡ)、转录因子CTCF和HMGA1的CUT&Tag实验,并利用现有的乙醇沉淀、片段分选(solid-phase reversible immobilization,SPRI)磁珠回收和直接PCR法,以及本研究建立的srCUT&Tag方法对产物进行回收。结果表明,从整体上看,SPRI磁珠回收和srCUT&Tag方法着较高的回收效率,而乙醇沉淀法则回收效率低下。在全部4种CUT&Tag产物回收过程中,SPRI磁珠回收均会损失大部分小于150 bp的产物片段。在CTCF和HMGA1 CUT&Tag产物的回收中,直接PCR法则损失了大部分大于300 bp的片段并与其他回收方法的结果有较大的差别。因此,srCUT&Tag能够比其他三种回收方法提供更多更完整的测序信息。综上所述,新建立srCUT&Tag回收方法相比现有的CUT&Tag产物回收方法能提高建库效率并得到更好的数据质量,为表观遗传学研究提供了更好的技术选择。     

磁珠富集法筛选大弹涂鱼CA微卫星序列

以大弹涂鱼(Boleophthalmus pectinirostris)基因组DNApool为材料,构建基因组PCR文库,采用链霉亲和素包被的磁珠富集法筛选目的片段.目的片段经克隆、测序验证后获得大弹涂鱼微卫星序列.在筛选的409个菌落中共获得209个阳性克隆,其中具有144个微卫星序列(GenBank登录号为HQ852252 - HQ852395),除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有117条.     

以gfp为报告基因的肺炎链球菌启动子诱捕文库的构建与分析

首先构建一个以gfp(green fluorescence protein)为报告基因的自杀质粒pEVP3-SDGFP,将肺炎链球菌基因组DNA的随机酶切片段(200bp～800 bp)克隆到该质粒gfp基因上游的多克隆位点,得到约58000个含有肺炎链球茵基因组DNA随机酶切片段的重组子,提取质粒即为质粒库,该库大约覆盖肺炎链球菌基因组全长的5倍,插入率达到90%以上,且有较强的随机性,质量较高.将该质粒库转化入肺炎链球菌TIGR4菌株,带有随机片段的报告质粒通过同源重组的方式将gfp基因融合于细菌染色体上该随机片段之后,利用质粒的抗生素抗性基因筛选出重组菌株,从而构建出相应的菌株库,共获得包含约500000个肺炎链球菌转化子的菌株库,经体内、外实验表明,其包含插入了S.pn体内、外表达基因片段的细菌,可以报告特定条件下的基因表达,并可通过流式细胞仪识别、分选.该文库的构建为进一步利用差异荧光诱导技术筛选肺炎链球菌体内诱导基因奠定了基础.     

单染色体靶向富集方法的建立

靶向富集技术具有良好的均一性、特异性和灵敏性,只需较低的测序覆盖度即可满足后续的分析。该技术被广泛应用于特定位点重测序和染色体构象等研究。为了靶向富集测序文库中特定的染色体DNA片段,本研究以21号染色体为例,采用人胚肝二倍体细胞CCC-HEL-1建立了一种单染色体靶向富集方法。具体步骤如下：（1）首先将人正常核型二倍体细胞同步化到G2/M 期,制备染色体悬液;然后流式分选出21号单染色体。（2）采用多重退火环形扩增技术（MALBAC）对21号染色体DNA进行扩增;用扩增的DNA产物构建文库。（3）利用体外转录合成、生物素标记的RNA探针与文库进行液相杂交,捕获并验证捕获效率。荧光定量PCR检测结果证明,特定的21号染色体呈上百倍的富集,而靶标文库则富集了近60 倍。结果提示,特异单染色体靶向富集方法成功建立。该法不仅可实现任一单染色体的靶向富集,用于单条染色体功能组学研究,而且还可能用于查找特定疾病相关的染色体异常（如DNA突变、异位、重复及倒置等）,具有一定的实用意义。     

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

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...     

Solid-Phase Nested Deletion: A New Subcloning-less Method for Generating Nested Deletions

We have developed a new subcloning-less method for generatingnested deletions which we have termed Solid-Phase Nested Deletion.The basic procedure for this method is as follows. The targetDNA fragment is cloned in the multiple cloning site of a cloningvector, pUC or its derivatives, and amplified by PCR using aset of primers, one of which is 5'-biotinylated. The amplifiedDNA is partially digested by a restriction enzyme with a 4-baserecognition sequence. The digested DNA is ligated with a syntheticadapter DNA. Monodiverse beads coupled with streptavidin (Dynabeads^TMM-280 streptavidin) are added to the mixture and the biotinylatedDNA fragments are separated by applying magnetic field. Theunidirectionally deleted DNA fragments are recovered by PCRfrom the magnetic beads, and size-fractionated by agarose gelelectrophoresis. The DNA fragments are amplified by PCR andused for sequencing. We demonstrate the potential of this methodusinga 4878-bp EcoRI fragment of phage DNA.     

Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).     

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。     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.     

巴氏蘑菇微卫星序列的分离及其对Co60辐射诱变菌株的鉴别

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。     

丁坝对鱼类栖地的影响范围评估

利用磁珠富集法和5’锚定PCR法开发背瘤丽蚌的微卫星标记,将获得的多态性引物用于群体的遗传多态性分析,以期在比较两种开发微卫星标记方法的基础上同时获得一批有用的微卫星引物。从磁珠富集法获得的微卫星序列阳性克隆率为69.2%,重复次数超过10的占总数的70.2%,从设计的28对引物中筛选得到多态性引物11对,开发效率为39.3%。这11对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为4～13,观测杂合度、期望杂合度范围分别为0.205～0.738、0.566～0.839。而5’锚定PCR法获得的微卫星序列阳性克隆率为97.8%,重复次数超过10的占总数的24.7%,从设计的56对引物中筛选得到多态性引物19对,开发效率为30.4%。这19对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为3～10,观测杂合度、期望杂合度范围分别为0.208～0.894、0.431～0.896。实验结果表明,磁珠富集法所获微卫星序列质量高,开发微卫星标记效率较高;而5’锚定PCR法实验操作更简便,所获得的引物遗传多样性指数更高。两种方法开发的引物均可用于背瘤丽蚌和近缘种的野生种质资源遗传多样性研究。     

A Modified Enrichment Method to Construct Microsatellite Library from Plateau Pika Genome （Ochotona curzoniae）

A microsatellite-enriched library of plateau pika(Ochotona curzoniae)was constructed according to the strong affinity between biotin and streptavidin.Firstly,genomic DNA was fragmented by ultrasonication,which is a major improvement over traditional methods.Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes,and then were subjected to streptavidin-coated magnetic beads.PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites.Ligation and transformation were carried out by using the pGEM-T Vector System Ⅰ and Escherichia coli DH10B competent cells.Sequencing results showed that 80.2% of clones contained microsatellite repeat motif.Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly,composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions.This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster(Cricetulus griseus)and small abalone[Haliotis diversicolor(Reeve)]with high rates of positive clones,demonstrating its feasibility and stability.