PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.     

红曲菌cDNA消减文库的构建

应用抑制性消减杂交技术,构建红曲菌产桔霉素和不产桔霉素差异表达的cDNA消减文库.分别从产桔霉素和不产桔霉素的红曲菌丝体中提取mRNA,依次合成单链和双链cDNA,经酶切成大小为250～750bp的片断,将产桔霉素的cDNA分为两组,分别与两种不同的接头连接,再与不产桔霉素的红曲菌的cDNA进行两次消减杂交及两次抑制性PCR后,将产物与T/A载体连接构建成功cDNA消减文库,并转染大肠杆菌进行文库扩增,文库扩增后得到283个克隆,经PCR法快速测定,均得到250～750bp的插入片断.所构建的红曲菌cDNA消减文库为进一步筛选红曲菌中与产桔霉素性状相关的基因奠定了基础.     

An improved method for subtractive cloning of differentially expressed genes in higher plants by protective exonuclease digestion and discriminating PCR amplification

An improved method for subtractive cloning with enhanced efficiency was developed by modifying the enzymatic degrading subtraction. The thionucleotide-modified tester cDNA fragments under control of one linker-primer were hybridized with excess driver cDNA fragments flanked by the other distinct linker-primer. After selective digestion of incompletely protected tester/driver and of unprotected driver/driver molecules with exonuclease III and VII, the protected tester/tester reassociates due to thionucleotides were exclusively amplified by PCR with the tester-cDNA-specific primer. The subtractively enriched target cDNA fragments, showing distinct bands in an agarose gel, were inserted into pUC19, and random colonies with inserts were screened by Northern hybridization to tester and driver RNA. Four distinct clones were confirmed to be up-regulated by the withdrawal of potassium from the nutrient solution of seedling barley growing hydroponically. The original protocol generated only smeared amplicons due to non-selective PCR amplification of the hybridized cDNA mixture including remains of undigested driver cDNA.Abbreviations EDS Enzymatic degrading subtraction - SET Subtractively enriched target     

PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism

A PCR-based positive hybridization (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of Anabaena circinalis, a cyanobacterium able to produce saxitoxin (STX). The PPH technique is based on the same principles of suppression subtractive hybridization (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridized at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A.circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed the recovery of DNA libraries that were mainly specific for each of the two STX-producing strains used. Several candidate sequences were found by PPH to be in common between both the STX-producing testers. The PPH technique performed using unsubtracted genomic libraries proved to be a powerful tool to identify DNA sequences possibly transferred laterally between two cyanobacterial strains that may be candidate(s) in STX biosynthesis. The approach presented in this study represents a novel and valid tool to study the genetic basis for secondary metabolite production in microorganisms.     

Construction and mapping of safflower chloroplast DNA recombinants and location of selected gene markers

Summary DNA was isolated and purified from chloroplasts of safflower (Carthamus tinctorius L.), digested with HindIII restriction endonuclease, and ligated into the HindIII site of the plasmid pUC9. Recombinant DNAs were isolated from ampicillin resistant white colonies which grew in the presence of the appropriate indicator, digested with HindIII, and then identified by comparison of agarose gel electrophoretic mobilities. HindIII digests of chloroplast DNA were used as a standard. Such recombinants were radiolabeled and hybridized with Southern blots of PstI, SalI, KpnI, and HindIII single and double digests of safflower chloroplast DNA. A physical map was subsequently generated showing the location of each recombinant on the circular plastid genome. Recombinants containing heterologous chloroplast gene markers from spinach or Euglena were also radiolabeled and mapped. The relative mapping positions of these genes are in good agreement with those which have previously been published for spinach and several other higher plants.     

水稻线粒体26S rRNA基因在大肠杆菌JM101中的克隆及分子鉴定

用BT型水稻喜峰A黄化苗为材料,按本实验过去报道经修改后的方法提取线粒体DNA,经EcoR1完全酶切后,随机克隆到pUC19载体上,转化大肠杆菌,在含氨苄青霉素(50μg/m1)和X-gal的LB固体平板上筛选白色转化子。随机提取重组子DNA,以玉米26S rRNA基因为探针,经Southern分子杂交鉴定,一个插入1.3kb水稻线粒体DNA片段的重组质粒杂交结果为阳性,并将这个含有26S rRNA基因片段的重组质粒命名为pXMT1。     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

Cloning of deleted sequences (CODE): A genomic subtraction method for enriching and cloning deleted sequences.

The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.     

KpnI families of long, interspersed repetitive DNAs in human and other primate genomes.

KpnI restriction of DNAs from all anthropoid primates studied releases a conspicuous series of segments representing families of long, interspersed repetitive DNAs termed here the KpnI 1.2, 1.5, 1.8 and 1.9 kb families. Human KpnI 1.2 to 1.9 kb segments representative of these families were isolated and separately cloned in the KpnI site of a plasmid pBK5, specially constructed for this purpose. The KpnI clones did not cross-hybridize with cloned, primate alphoid sequences, suggesting that the KpnI families represent sequences separate and distinct from the alphoid DNAs. Secondary restriction analyses of cloned KpnI segments demonstrated microheterogeneity among individual members within the same KpnI family. Autoradiograms of capuchin monkey, AGM and human DNA cleaved with HaeIII, AluI or RsaI and hybridized to various cloned human KpnI sequences demonstrated a remarkable conservatism and relative simplicity in the organization of the KpnI families in the genomes of these widely divergent primates. The KpnI 1.2 kb and 1.5 kb families occur in high frequency (15%) among all plaques in two recombinant human genome libraries. Evidence is presented suggesting that the bulk of the KpnI families occur in the genome as clusters or congeries of higher molecular weight segments (greater than 2 kb) containing sequences homologous to the low molecular weight segments (1.2 to 1.9 kb).     

Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

Leptospirosis is a globally important zoonotic diseasecaused by the pathogenic species of the spirochete genus,Leptospira including L. interrogans, L. kirschneri, L.noguchii, L. borgpetersenii, L. santarosai, L. weilii andetc. [1]. Pathogenic leptospires …     

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.     

Microdissection of M chromosome in Vicia faba and its library construction]

Microdissection and microcloning technique was employed to construct the library of M chromosome in Vicia faba. The M chromosomes were microdissected with a micromanipulator and were put into a 0.5 ml Eppendorf tube, then digested with Sau3A. Sau3A linker adaptors were ligated to the end of chromosome DNA fragments, and two rounds of PCR were carried out with one chain of linker adaptor as the primer. The PCR products ranged in size from 300 base pair (bp) to 3000 bp with predominant fragments from 500 bp to 1500 bp. Southern hybridization analysis confirmed that PCR products originated from Vicia faba genome. The second round PCR products were cloned and about 102,000 recombinants were obtained. 118 recombinants were selected randomly for analysis. The inserts ranged in size from 150 bp to 3000 bp with an average of 690 bp. Dot blot was carried out for 100 clones with DIG labeled Vicia faba genome DNA as probes. The result revealed that 51% were low and unique copy sequences, 49% were repetitive sequences. M chromosome DNA library has not been reported before.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.     

Restriction endonuclease cleavage site map of safflower (Carthamus tinctorius L.) chloroplast DNA

Summary The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with ³²P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular and to contain approximately 151 kbp. In common with many other higher plant chloroplast DNAs a sequence of about 25 kbp is repeated in an inverted orientation. The small and large single copy regions separating the two repeated segments contain about 20 kbp and 81 kbp, respectively. The rRNA structural genes were also mapped by Southern blot hybridization and are co-linear with several other plant species.     

用衔接头PCR克隆新的胡萝卜Ⅱ型转化酶基因启动子

 为克隆新的胡萝卜 型转化酶基因启动子 ,将胡萝卜基因组 DNA分别用 Pvu 、Eco R 、Dra 和 Sma 酶切 ,酶切片段与一特殊的衔接头连接 .取连接产物作模板 ,以衔接头引物和基因特异引物做 PCR,得到的主要 PCR产物分别为 3.4kb、1 .3kb、0 .4kb和 0 .6kb.将 Eco R -衔接头体系的 PCR产物克隆和测序 ,并将其序列与 Gen Bank中的已知序列进行比较分析 ,发现了一个新的胡萝卜 型转化酶启动子序列 ,它含有类似于 TATA box和 CAAT box的元件 ,在启动子的远上游区域还含有多个 AT富含区 .该启动子的发现对于研究植物中糖代谢具有重要意义 .     

A simulation of subtractive hybridization.

Various strategies employed in genomic DNA cloning by subtractive hybridization have been examined by computer simulations, with the comparison between the predictions and the published results. The result shows that the efficiency of target sequence enrichment and the sensitivity to experimental conditions depend strongly on the enrichment strategy employed. The strategy selecting only tester/tester after hybridization can be very efficient to enrich targets. For successful target enrichment, however, the strategy requires a highly efficient subtraction method and proper hybridization conditions. The strategy also requires that the selected DNA be amplified by polymerase chain reaction (PCR) after each or each alternate subtraction. By contrast, the strategy selecting tester/tester plus single-stranded tester is less sensitive to various experimental factors, compared with the strategy selecting only tester/tester. However, it is not as efficient. With this strategy, the tester DNA selected may or may not be amplified by PCR before the next round. In the case of the strategy selecting single-stranded tester, the target DNA can be successfully enriched only when the selected DNA is directly used without PCR amplification in the next round. The strong features of existing methods can be combined to develop a protocol that is more efficient and more reliable.     

The Isolation of differentially expressed mRNA sequences by selective amplification via biotin and restriction-mediated enrichment

Molecular analysis of development frequently implies the isolation and characterization of genes with specific spatial and temporal expression patterns. Several methods have been developed to identify such DNA sequences. The most comprehensive technique involves the genomewide probing of DNA sequence microarrays with mRNA sequences. However, at present this technology is limited to the few organisms for which the entire genome has been sequenced. Here, we describe a subtractive hybridization technique, called selective amplification via biotin and restriction-mediated enrichment (SABRE), which allows the selective amplification of cDNA fragments representing differentially expressed mRNA species. The method involves the competitive hybridization of an excess of driver cDNA fragments (D) to a trace of tester cDNA fragments (T), and the subsequent purification of tester homohybrids (in which both strands are contributed by the tester cDNA). After competitive hybridization, cDNA fragments that are more abundant in the tester than in the driver are enriched in the tester homohybrids. However, as the fraction of tester homohybrids is very small [T(2)/(D + T)(2)], their purification requires highly efficient procedures. In SABRE, the isolation of tester homohybrids is afforded by a combination of three successive steps: removal of biotinylated terminal sequences from most of the heterohybrids by S1 nuclease digestion, capture of biotinylated hybrids with streptavidin-coated paramagnetic beads, and specific release of homohybrids from the beads by restriction nuclease digestion. If several rounds of SABRE selection are performed in series, even relatively rare differentially expressed mRNA sequences may result in the production of predominant cDNA fragments in the final tester homohybrid population.     

Development of a fast DNA-DNA hybridization method based on melting profiles in microplates

DNA-DNA hybridization is still the “gold standard” for the genotypic delineation of bacterial species. However, it is not widely used because traditional DNA-DNA hybridization techniques are rather time-consuming and not easy to perform in routine laboratories. In the present study, DNA of reference strains was digested with Sau3A, ligated with linker oligonucleotides S1/2 and in vitro amplified. The amplified DNA fragments were immobilized on MaxiSorb 96-well plates. DNA isolated from target strains was also digested with Sau3A, ligated with linker oligonuleotides P1/2 and in vitro amplified in the presence of digoxygenin modified dUTP. The labeled amplificate was hybridized to the immobilized reference DNA under isothermal conditions. Thermal denaturation curves of the DNA-DNA hybrids were obtained by using washing solutions of increasing stringency. Remaining hybrids were colorimetrically detected with anti-digoxygenin-horseradish peroxidase anti-bodies. The new method was validated with strains of the genus Pedioccocus for which DNA-DNA similarities have also been determined by the filter hybridization method. In addition, DNA-DNA hybridizations were performed with genotypically defined Enterobacter species.