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     

Second-generation approach to the construction of yeast artificial-chromosome libraries

We describe an improved method for construction of yeast artificial-chromosome (YAC) libraries that contain large inserts of foreign DNA. The procedure consists of seven steps: (i) preparation of human DNA in agarose beads; (ii) partial digestion of the DNA with EcoRI; (iii) electrophoretic elimination of the smaller partial-digest fragments; (iv) ligation of the EcoRI fragments with vector arms in molten agarose; (v) hydrolysis of agarose with agarase; (vi) fractionation of the recombinant molecules by sucrose-gradient centrifugation; and (vii) transformation of yeast. More than 7000 colonies were obtained starting with 15 micrograms of human DNA, which was fractionated on a single sucrose gradient. The average size of these YACs was approximately 380 kb. It is estimated that the total length of human DNA present in the clones corresponds to 80% of the length of the human haploid genome. The results of screening the clones for a number of single-copy genes indicate that the clones reflect a nearly random sampling of the human genome. The efficiency of the cloning is sufficient to support the construction of multihit libraries for the human genome or for the genomes of other higher organisms.     

Preparation of high molecular weight plant DNA and its use for artificial chromosome construction

Summary The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analyzed. Conditions have been established for the obtainment of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension. Restriction fragments were obtained by partial and total digestion with different endonucleases, and separated by pulsed-field gel electrophoresis. Ligation of partially EcoRI-digested DNA (range 30–300 kbp) followed by transformation of yeast spheroplasts gave rise to YACs with an average size of 60 kbp. The introduction of a DNA size-selection step before ligation led to production of YACs in the range of 100–200 kbp. Clones of up to 460 kbp were obtained by blunt-end ligation of pre-selected unrestricted DNA.Abbreviations 2,4-D 2,4-dichloro phenoxyacetic acid - 6BAP 6-benzylaninopurine - BFP bovine serum albumin 0.1%, Ficoll 400 0.1%; polyvinylpyrrolidone 0.1% - CHEF clumped homogeneous electric field - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - HMW high molecular weight - km kanamycin - LMP agarose low melting point agarose - MS Murashige and Skoog mediun - npt neomycin phosphotransferase - PEG polyethyleneglycol - PFGE pulsed-field gel electrophoresis - RFLP restriction fragrant lenght polymorphism - SDS sodium dodecyl sulphate - SSC sodium chloride 150 mM, sodiun citrate 15 nM, pH 7 - TAE TRIS-Acetate pH 8 40 mM, EDTA 2 mM - TE TRIS-HCl pH 8 10 mM, EDTA 1 mM - YAC yeast artificial chromosome     

An efficient method for purification of PCR products for sequencing

A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%-0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequencing reaction. This method is simple and labor efficient, provides high quality sequences at a low cost, and bypasses problems with impure PCR products. This technique has been used for single nucleotide polymorphism (SNP) discovery in Populus angustifolia trees.     

Cloneless genomic DNA analysis: an efficient and simple methods for de novo genomic sequencing projects and gap filling

The utility of using genomic DNA directly in agarose, i.e. cloneless libraries, in place of large clone libraries, radiation hybrid panels, or chromosome dissection was demonstrated. The advantage of the cloneless library approach is that, in principle, a targeted genomic resource can be developed rapidly for any genomic region using any genomic DNA sample. Here, a human chromosome 20 Not I fragment library was generated by slicing a pulsed field gel lane containing fractionating Not I cleaved DNA from a monosomic hybrid cell line into 2 mm pieces. A reliable PCR method using agarose embedded DNA was developed. InterAlu PCR generated unique patterns of products from adjacent slices (e.g. fractions). Further, the specificity of the interAlu products was demonstrated by FISH analysis and in other hybridization experiments to arrayed interAlu products. STS content mapping was used to order the fractions and also demonstrate the unique content of the library fractions.     

The only domain which distinguishes Epstein-Barr virus latent membrane protein 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro; LMP2A is therefore nonessential.

Using second-site homologous recombination, Epstein-Barr virus (EBV) recombinants were constructed which carry an LMP2A mutation terminating translation at codon 19. Despite the absence of LMP2A or LMP2A cross-reactive protein, the recombinants were able to initiate and maintain primary B-lymphocyte growth transformation in vitro. EBNA1, EBNA2, and LMP1 expression was unaffected by the LMP2A mutation. The LMP2A mutant recombinant EBV-infected lymphoblastoid cell lines (LCLs) were identical to wild-type recombinant EBV-infected control LCLs with respect to initial outgrowth, subsequent growth, sensitivity to limiting cell dilution, sensitivity to low serum, and growth in soft agarose. The permissivity of LCLs for lytic EBV infection and virus replication was also unaffected by the LMP2A mutation.     

Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.     

Epstein-Barr virus latent membrane protein 1 represses DNA repair through the PI3K/Akt/FOXO3a pathway in human epithelial cells

Latent membrane protein 1 (LMP1), an Epstein-Barr virus (EBV) oncoprotein, mimics a constitutively activated tumor necrosis factor receptor and activates various signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt. LMP1 is essential for EBV-mediated B-cell transformation and is sufficient to transform several cell lines. Cellular transformation has been associated strongly with genomic instability, while DNA repair plays an important role in maintaining genomic stability. Previously, we have shown that LMP1 represses DNA repair by the C-terminal activating region 1 (CTAR1) in human epithelial cells. In the present study, we demonstrate that the PI3K/Akt pathway is required for LMP1-mediated repression of DNA repair. Through the LMP1/PI3K/Akt pathway, FOXO3a, which can induce DNA repair, is inactivated because of phosphorylation and relocalization. Expression of a constitutively active FOXO3a mutant can rescue LMP1-mediated repression of DNA repair. Furthermore, LMP1 can decrease the expression of DNA damage-binding protein 1 (DDB1), which functions in nucleotide excision repair, through the PI3K/Akt/FOXO3a pathway. LMP1-mediated repression of DNA repair is restored by DDB1, although only partially. These results suggest that LMP1 triggers the PI3K/Akt pathway to inactivate FOXO3a and decrease DDB1, which can lead to repression of DNA repair and may contribute to genomic instability in human epithelial cells.     

Reduced Expression of the Antigen Processing Machinery Components TAP2, LMP2, and LMP7 in Tonsillar and Base of Tongue Cancer and Implications for Clinical Outcome

OBJECTIVES: Patients with human papillomavirus (HPV)–positive tonsillar squamous cell carcinoma (TSCC) and base of tongue squamous cell carcinoma (BOTSCC) have a better clinical outcome than those with corresponding HPV-negative tumors. Moreover, there is a strong positive correlation between absent/low as opposed to strong HLA class I expression and favorable clinical outcome for HPV-positive tumors, while the reverse applies to HPV-negative tumors. The expression of the antigen processing machinery (APM) components TAP1, TAP2, LMP2, and LMP7 in these tumors in relation to HPV status, HLA class I expression, each other, and clinical outcome was therefore investigated. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded TSCC and BOTSCC, derived from 151 patients and previously analyzed for HPV DNA, HLA class I, and LMP10 expression were stained by immunohistochemistry for TAP1, TAP2, LMP2, and LMP7. RESULTS: Absent/low TAP2, LMP2, and LMP7 expression, similar to HLA class I and LMP10, was common in TSCC and BOTSCC, irrespective of HPV status. Expression of TAP1 and TAP2 was correlated, as was LMP2 to LMP7. LMP2 and LMP7 expression was also associated to HLA class I expression. Moreover, absence of LMP7 was linked to increased disease-free survival in both HPV-positive and HPV-negative cases. CONCLUSION: Reduced expression of TAP2, LMP2, and LMP7 was frequent in TSCC and BOTSCC and their expression as well as that of TAP1 was often interrelated. Furthermore, low LMP7 expression correlated to better clinical outcome and may, together with HPV status, potentially be used for prediction of treatment response.     

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 and characterization of human DNA in agarose block

Classic phenol-chloroform extraction of human DNA results in substantial shearing and low yield. Because DNA analysis in human genetic disorders needs relatively intact DNA, we modified the existing method and systematically analyzed the human DNA isolated from HeLa cells, leukocytes, amniocytes, and fibroblasts in agarose block and compared the results to those obtained by conventional phenol method. Our results showed that DNA isolated by the agarose method was higher in molecular weight, with minimal shearing as compared to the phenol method. Yield of DNA from the agarose method was substantially higher, almost twice that obtained by the phenol method. Restriction enzyme digestion of DNA from the agarose method indicated the usefulness of this DNA for restriction fragment length polymorphism (RFLP) analysis without further purification. DNA obtained by the agarose method was found to be more resistant to thermal degradation and more stable on long-term storage than that of phenol-extracted DNA.     

Epstein-Barr virus-specific cytotoxic T-cell recognition of transfectants expressing the virus-coded latent membrane protein LMP

Cytotoxic T cells from Epstein-Barr virus (EBV)-immune individuals specifically kill EBV-transformed B cells from HLA class I antigen-matched donors even though the latently infected cells express only a restricted set of virus genes. The virus-induced target antigens recognized by these immune T cells have not been identified. In our experiments, EBV DNA sequences encoding the virus latent gene products Epstein-Barr nuclear antigen (EBNA)1, EBNA 2, and EBNA-LP and the latent membrane protein (LMP) were individually expressed in a virus-negative human B-lymphoma cell line, Louckes. Transfected clones expressing LMP were killed by EBV-specific cytotoxic T-cell preparations from each of three virus-immune donors HLA matched with Louckes through HLA-A2, B44 antigens; control transfectants or clones expressing one of the EBNA proteins were not recognized. Expression of LMP in a second virus-negative B-cell line, BL41, sensitized these cells to EBV-specific cytolysis restricted through the HLA-A11 antigen. To distinguish between the viral protein and an induced human B-cell activation antigen as the target for T-cell recognition, LMP was then expressed in a murine mastocytoma cell line, P815-A11-restricted human T cells. The LMP-expressing P815-A11 transfectants were susceptible to lysis by EBV-specific cytotoxic T cells from three HLA-A11-positive individuals. Both Louckes and P815-A11 cells were also transfected with constructs capable of encoding a truncated form of LMP (Tr-LMP) which lacks the N-terminal 128 amino acids of the full-length protein. Tr-LMP-expressing transfectants were not recognized by the above T-cell preparations. The results suggest that LMP, and, in particular, epitopes derived from the N-terminal region of the protein, provides one of the target antigens for the EBV-induced human cytotoxic T-cell response.     

Electrophoretic characterization of chromosomal DNA from two microsporidia

Spores of two microsporidia, Nosema pyrausta (from the European corn borer, Ostrinia nubilalis) and N. furnacalis (from the Asian corn borer, O. furnacalis) were harvested from laboratory-reared O. nubilalis caterpillars and purified by centrifugation through Percoll. Conditions permitting in vitro germination were defined for both species and found to be different. N. pyrausta spores were incubated in 0.1 N KOH for 30 min, recovered by centrifugation, and resuspended in 1 ml of an equal mixture of 1% low melting point (LMP) agarose and L-15B medium at 37 degrees C to induce germination. N. furnacalis spores were first washed in 10 mM Na2EDTA in 1 mM Tris base, pH 7.5, exposed to 0.01 N KOH in 0.17 M KCl for 30 min, centrifuged, and germinated in 1 ml of an equal mixture of 1% LMP agarose and 0.17 M KCl in 10 mM Na2EDTA (pH 8), at 37 degrees C. Eighty to 90% of the spores of each species germinated. Germinated spores were pipetted into a casting mold. Before electrophoresis, agarose blocks were incubated 48 hr at 50 degrees C in 10 mM Tris base/100 mM Na2EDTA, pH 7.8, with 1 mg/ml proteinase K and 1% N-laurylsarcosine to release the chromosomal DNA from sporoplasms. After pulsed-field electrophoresis, ethidium bromide staining revealed 13 chromosomal bands ranging in size from 1390- to 440-kb pairs and 1360- to 440-kb pairs in N. pyrausta and N. furnacalis, respectively. The difference in size estimates of corresponding chromosomes in the two species was not more than 60-kb pairs.     

Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci

Two bacterial artificial chromosome (BAC) libraries were constructed from an inbred line derived from a cultivar of cucumber (Cucumis sativus L.). Intact nuclei were isolated and embedded in agarose plugs, and high-molecular-weight DNA was subsequently partially digested with BamHI or EcoRI. Ligation of double size-selected DNA fragments with the pECBAC1 vector yielded two libraries containing 23,040 BamHI and 18,432 EcoRI clones. The average BamHI and EcoRI insert sizes were estimated to be 107.0 kb and 100.8 kb, respectively, and BAC clones lacking inserts were 1.3% and 14.5% in the BamHI and EcoRI libraries, respectively. The two libraries together represent approximately 10.8 haploid cucumber genomes. Hybridization with a C₀t-1 DNA probe revealed that approximately 36% of BAC clones likely carried repetitive sequence-enriched DNA. The frequencies of BAC clones that carry chloroplast or mitochondrial DNA range from 0.20% to 0.47%. Four sequence-characterized amplified region (SCAR), four simple sequence repeat, and an randomly amplified polymorphic DNA marker linked with yield component quantitative trait loci were used either as probes to hybridize high-density colony filters prepared from both libraries or as primers to screen an ordered array of pooled BAC DNA prepared from the BamHI library. Positive BAC clones were identified in predicted numbers, as screening by polymerase chain reaction amplification effectively overcame the problems associated with an overabundance of positives from hybridization with two SCAR markers. The BAC clones identified herein that are linked to the de (determinate habit) and F (gynoecy) locus will be useful for positional cloning of these economically important genes. These BAC libraries will also facilitate physical mapping of the cucumber genome and comparative genome analyses with other plant species.     

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.     

水稻二化螟和三化螟基因组ddRADseq文库的构建

本文介绍了构建水稻二化螟和三化螟"双酶切限制性酶切位点关联DNA测序"(Double digest restrictionsite associated DNA sequencing,ddRADseq)文库的方法。利用安捷伦2100生物分析仪对4种单酶切及2种双酶切的酶切产物片段大小及分布范围进行分析,筛选出Mlu C I和Nla III两种限制性内切酶组合对螟虫基因组DNA进行酶切。酶切后的DNA片段两端连接上特定的P1、P2接头后,用Pippin Prep回收大小为285-435 bp的DNA片段。通过PCR扩增进行文库的富集并引入index序列。构建好的ddRADseq文库用琼脂糖凝胶电泳和生物分析仪进行质量检测。本方法所构建的文库DNA片段长度、分布和摩尔浓度能够达到Illumina平台测序的技术要求。本研究证实了利用Mlu C I和Nla III组合酶切构建水稻螟虫基因组ddRADseq文库的可行性,为在水稻螟虫中利用ddRADseq技术开展生物地理学、种群遗传学和系统发育重建等方面的研究奠定基础。     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.