Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

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     

Method for quick DNA barcode reference library construction

DNA barcoding has become one of the most important techniques in plant species identification. Successful application of this technology is dependent on the availability of reference database of high species coverage. Unfortunately, there are experimental and data processing challenges to construct such a library within a short time. Here, we present our solutions to these challenges. We sequenced six conventional DNA barcode fragments (ITS1, ITS2, matK1, matK2, rbcL1, and rbcL2) of 380 flowering plants on next‐generation sequencing (NGS) platforms (Illumina Hiseq 2500 and Ion Torrent S5) and the Sanger sequencing platform. After comparing the sequencing depths, read lengths, base qualities, and base accuracies, we conclude that Illumina Hiseq2500 PE250 run is suitable for conventional DNA barcoding. We developed a new “Cotu” method to create consensus sequences from NGS reads for longer output sequences and more reliable bases than the other three methods. Step‐by‐step instructions to our method are provided. By using high‐throughput machines (PCR and NGS), labeling PCR, and the Cotu method, it is possible to significantly reduce the cost and labor investments for DNA barcoding. A regional or even global DNA barcoding reference library with high species coverage is likely to be constructed in a few years.     

不同微生物的单光束激光陷阱操纵

本文报导了分别采用Ｈｅ－Ｎｅ和Ａｒ＾＋激光器与光不显微镜构成的单光束激光陷阱操纵酵母菌、青霉等不同微生物的实验观察结果,讨论了操纵条件。研究表明：用单光束高会聚激光产生的梯度力操纵微生物体,其有效作用力的大小不仅与激光功率、波长、束腰半径和光束会聚角有关,还与微生物的大小、吸收系数、菌龄及培养方法等因素有关。     

QTL mapping of protein content in rice using single chromosome segment substitution lines

Protein content (PC) is an important component of rice nutritional quality. In order to better understand the genetic basis of this trait and increase related breeding efficiency, 21 single chromosome segment substitution (SCSS) lines grown in four sites over two growing seasons (regarded as eight environments) were used to associate PC with particular chromosome segments. Segments from 15 chromosomes were found to contain quantitative trait loci (QTLs) for PC in at least one environment. These included segments from chromosome 3 and 8, in which QTLs for PC had not previously been identified. The segment of chromosome 8 in CSSL-48 had the largest positive effect across all environments. The interaction between substitution and environment was highly significant. Some substitutions had large effects in one environment, but no effect in another (i.e. CSSL-08 and CSSL-17), while some substitutions significantly increased PC in one environment but decreased it in another (i.e. CSSL-41 and CSSL-43). By biplot and clustering analysis, the eight environments were grouped into two contrasting environment types, that is, Hainan and Jiangsu. The segment of chromosome 8 in CSSL-48 had PC-enhancing QTLs in both of the environment types. The segments in CSSL-34 had QTLs which increase PC in the Jiangsu environment but have no effect in the Hainan environment. For enhancing PC, CSSL-48 could be explored in breeding for wide adaptation across all environments, while CSSL-12, CSSL-14, CSSL-17, CSSL-41 and CSSL-43, and that in CSSL-34 could be explored in breeding for specific adaptation to the Hainan and Jiangsu environments, respectively. Near isogenic lines are under development to validate the QTLs with large effects in a range of genetic backgrounds relevant to Jiangsu and Hainan breeding programs. Secondary mapping populations are also being developed for further localising the responsible QTLs in CSSL-14, CSSL-34 and CSSL-48.     

Preparation of a genomic library using a TA vector

An efficient and simple method for constructing a genomic DNA library is presented using a TA cloning vector. It is based on sonication cleavage of genomic DNA, blunting of the fragment ends with mung bean nuclease, and addition of a single 3'-deoxyadenylate with Taq DNA polymerase, followed by ligation with a TA vector. This method is useful for improving the quality of genomic libraries for organisms whose genomic DNA is not well digested with restriction enzymes owing to the presence of polysaccharides and/or DNA methylation.     

Construction of poplar (Populus tremula) chromosome 1-specific DNA library by using a microdissection technique

A method for single-chromosome microdissection and microcloning was established in forest plants using poplar (Populus tremula) as a model. By use of meristematic cell division in root tip and the wall degradation hypotonic method, well-spread poplar metaphase chromosome spreads showing low contamination were quickly prepared and fitted for chromosome microdissection. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with a fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by theSau3A linker adaptor-mediated PCR technique, by which 200- to 3000-bp smear DNA fragments were obtained. Southern hybridization results showed that the PCR products from the single poplar chromosome were homogeneous with poplar genomic DNA, indicating that DNA from the single chromosome has been successfully amplified. Next, the second-round PCR products from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. About 3×10⁵ recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230–2200 bp, with an average of 800 bp. Therefore, this research suggests that microdissection and microcloning of single small chromosomes in forest plants is feasible.     

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.     

Microdissection of human chromosomes by a laser microbeam

A laser microbeam apparatus, based on an excimer laser pumped dye laser is used to microdissect human chromosomes and to isolate a single chromosome slice.     

Analysis of complex Y chromosome aberrations using a single DNA probe (Y-367)

A specific cloned DNA sequence (Y-367) detects at least four loci in the euchromatic long arm and in the short arm of the human Y chromosome. Deletion mapping assigns one locus to the distal euchromatic long arm, another to a region close to the centromere on either Yq or Yp, and two additional loci to the Y short arm. Y-367 may thus be used for the rapid screening of even complex Y chromosome aberrations. This is exemplified in a 45,X male with Y chromosome material on the long arm of chromosome 10 by the detection of an inversion of a portion of Yp and by the confirmation of duplications and deletions in two individuals with duplications of part of the Y chromosome.     

Regional mapping of unique DNA sequences from human chromosome 3 derived from a flow-sorted chromosome library

Eight single-copy DNA probes specific for human chromosome 3 were isolated by screening a human chromosome 3-derived genomic library. Southern blot analyses of DNAs isolated from a panel of somatic cell hybrids allowed us to regionally assign all probes to subregions on chromosome 3. Three clones were localized to the short arm of chromosome 3 (3p21----pter), two to the long arm (3q21----qter), and three to the 3q21----3p21 subregion. Six of these DNA sequences map to regions overlapping a segment of chromosome 3 (3p14----p23) frequently deleted in small cell lung cancer cells. Restriction fragment length polymorphism analyses indicate that at least three of the eight single-copy probes studies show MspI or BglII polymorphisms. This library is a useful source of chromosome 3-specific probes.     

染色体特异DNA文库的构建与鉴定

为构建人类21号染色体特异DNA文库, 以应用于人类遗传疾病的鉴定和研究, 文章采用循环温度梯度法溶解释放微分离的人外周血细胞21号染色体DNA, 将其进行简并寡核苷酸引物PCR(Degenerate oligo nucleotide primer-PCR, DOP-PCR)扩增后, 利用100~500 bp和500~2 000 bp分段回收纯化的两种不同片段大小的DOP-PCR产物构建染色体特异DNA文库, 并分别采用荧光原位杂交(Florescence in situ hybridization, FISH)和斑点杂交对DOP-PCR产物的来源和随机取样的文库克隆进行检测以评估所构建DNA文库的特异性。结果表明: 循环温度梯度法能有效溶解释放微分离的21号染色体DNA; 通过对DOP-PCR产物的分段回收纯化和克隆, 增加了大片段DNA的连接效率; 利用FISH技术和斑点杂交双重鉴定实验证明了文库的特异性, 从而构建了21号染色体特异的DNA文库, 并建立了构建染色体特异DNA文库及检测其特异性的方法, 为21号染色相关遗传疾病的鉴定和研究奠定了基础。     

Structural organization of alpha-satellite DNA in a single monkey chromosome

The organization of α-satellite sequences in a single monkey chromosome has been studied by restriction endonuclease analysis and molecular cloning. A somatic cell hybrid containing the monkey chromosome was isolated by cloning after fusion of the mouse L-cell line B82 (thymidine kinase minus) with primary African green monkey kidney cells and selective growth in HAT medium. Unlike the mouse cells, the hybrid cells contain DNA that hybridizes with the α-satellite DNA of the monkey. The presence of a single α-satellite containing monkey chromosome was demonstrated by Giemsa-11 staining and by the absence of both this chromosome and monkey α-satellite DNA sequences in cells after back-selection in bromodeoxyuridine. Hybridization of restriction endonuclease-digested hybrid cell DNA with a cloned segment of African green monkey α-satellite DNA showed distinctly different patterns from those observed with monkey total DNA. In particular, EcoRI and HaeIII restriction endonuclease sites are much more abundant in the satellite sequences in the thymidine kinase-carrying chromosome than they are in total satellite. A library of hybrid DNA was constructed in a λ bacteriophage. Analyses of purified recombinant phage that hybridized with α-satellite also indicated an abundance of EcoRI and HaeIII sites. Of nine phage studied in detail, no two showed identical distributions of the two restriction sites in the α-satellite sequences, suggesting the independent evolution of different domains within the single chromosome. These results indicate that the thymidine kinase-carrying chromosome contains distinct subsets (domains) of the α-satellite DNA of the whole monkey genome and further, that while the satellite sequence on the single chromosome is distinctive, it is also complex.     

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques

A well‐covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self‐primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four‐sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400–500 bp without bringing or inducing any sequence errors. About one‐third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well‐covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA‐labelled next‐generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality.     

Anonymous DNA probes to human chromosome 16 derived from a flow-purified library.

Anonymous DNA probes specific for human chromosome 16 were isolated from a flow-purified human chromosome 16 library. The library was constructed at the Lawrence Livermore National Laboratory. Twenty-nine clones containing a unique or low-copy DNA insert were isolated. Of these, six were assigned to chromosome 16 and regionally mapped and 12 were shown not to map to chromosome 16. One clone mapped to 16pter----16p13.1, one clone to 16p11.1----16q13, one clone to 16q13----16q22.1, and three clones to 16q22.1----16q24. An additional clone from the same library was mapped to 16q13----16q22.1.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome.

We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.