Strategies for optimizing DNA hybridization on surfaces

Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.     

Topology of syngameons

Syngameons are sets of species linked by interspecific hybridization. Common observations regarding the structure of syngameons are that hybridization propensity is not uniform across species and that patterns of hybridization are dominated by a few species. I use computer simulations to test these claims in naturally occurring syngameons selected from the literature and from personal observation. Natural syngameons, especially those involving plants, typically exhibit nonrandom structure: The first three order statistics for the number of hybrid partners and the variance in the number of hybrid partners are larger than chance alone would predict. The structure of two insect syngameons examined is not significantly different from random. To test a hypothesis that variation in hybridization propensity across species in natural syngameons is simply an artifact of hybridization opportunity, I examine the structure of four artificial syngameons (fertility relationships) produced by full diallel crosses. Three of four artificial syngameons exhibit nonrandom structure, as the observed variation in number of successful crosses is larger than chance alone would predict. In general, there are no significant results involving the order statistics. Finally, I discuss biogeographic, ecological, and phylogenetic hypotheses for variation in hybridization propensity across species in natural syngameons.     

水稻体细胞杂交研究进展

水稻是世界重要的粮食作物,全世界约有120个国家种植水稻。水稻的近缘或远缘种具有一些优良性状,如抗病、抗虫、抗逆等。将这些性状导入水稻,是科学家们所希望的,但因用普通杂交方法存在交配系统的不亲和性等难题而进展不大。随着组织培养技术的发展,特别是植物原生质体培养技术的日趋成熟,使得人类能够在细胞水平通过体细胞杂交方法实现遗传信息的重组。     

Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing ~(32)p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a ~(32)P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.     

Effects of various fluorochromes and competition between labeled oligonucleotides on their hybridization to oligonucleotides immobilized on biological microchips

Some technical tips are described on how to improve the discrimination between perfect and imperfect duplexes formed by hybridization of fluorescently labeled oligonucleotides to biological microchips. Model experiments were performed to assess the precision of the method. Effects of labeling on the efficiency of hybridization and some properties of competitive hybridization were studied using short synthetic oligonucleotides and three most popular fluorochromes as examples.     

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。     

Acute and Persistent Suppression of Preproenkephalin mRNA Expression in the Striatum Following Developmental Hypoxic-Ischemic Injury

Abstract: The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.     

Natural hybridization and introgression among sympatrically distributed Rhododendron species in Guizhou,China

Natural hybridizations occur among Rhododendron delavayi, R. decorum and R. irroratum, however, there was little study that had addressed the interbreeding behaviors when the three species grew in the same geographic areas. In this study, 37 accessions, containing the three species and the putative hybrids, were obtained from Baili Rhododendron Nature Reserve, Guizhou, China. Examinations of hybridization patterns with AFLP markers have led to a total of 107 diagnostic DNA fragments, which were analyzed with Principal Co-ordinate, Structure and NewHybirds analyses. The data confirmed that the existence of hybrids originated from the interbreeding between R. delavayi and R. irroratum in Baili Rhododendron Nature Reserve. R. decorum did not appear to be involved in the hybridization. Furthermore, most hybrids detected were a result of backcrossing, indicating that this hybrid differed from F1 dominant hybrids between R. delavayi and R. irroratum found in previous studies.     

普通小麦×大麦杂交后代中间材料的GISH及PAGE鉴定

利用基因组荧光原位杂交 (GISH)及种子贮藏蛋白聚丙烯酰胺凝胶电泳 (PAGE)对普通小麦×大麦杂交后代中间材料进行了鉴定分析。GISH结果表明 ,WBA984和WBA9812为二体小大麦异附加系 ,WBS0 2 15和WBS0 2 6 4为小大麦二体异代换系 ,WBT0 2 12 5和WBT0 2 183为端部易位系 ;种子贮藏蛋白PAGE分析表明 ,WBA9812和WBS0 2 6 4含有大麦特有的高分子量麦谷蛋白亚基和在γ区含有大麦特有的醇溶蛋白带型 ,WBA9812为大麦 5H附加系 ,WBS0 2 6 4为 1B/ 5H代换系 ,WBT0 2 12 5为 1BL/ 5HL端部易位系。     

Polyploidy in fishes: patterns and processes

Fishes are the most speciose group of vertebrates, with more than 24 000 species. They are characterized by great diversity in ecology, morphology, life history, behaviour and physiology. Here, the phylogenetic patterns of orders in which polyploidy has been recorded are considered, with special reference to patterns of species richness and hybridization: these orders include such phylogenetically diverse taxa as the Lepidosireniformes (lungfish) and the Perciformes (perch). Examples, predominantly drawn from the Cyprinidae and Salmonidae, are used to illustrate attributes of polyploidy in fishes. It is concluded (i) that polyploidy may have been of considerable importance in the evolution of fishes, and (ii) that fishes, with their diverse life histories, represent a useful model system with which to test theories relating to the origin and consequences of polyploidy that have been derived from work on plants.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 431–442.     

Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci

Abstract DNA-rRNA and DNA-DNA hybridization studies indicate that the classical pyogenic streptococci can be divided into five homology clusters. Based on these studies the term pyogenic streptococci should be confined to the first cluster consisting of serological groups A, A-variant, C, G ('large' colony, type II) and L.
Streptococci of serological groups B and M form the second cluster. The third cluster is composed of streptococci of serological groups R and S and serological groups U, V and P are found in the fourth cluster. The fifth cluster comprises strains of Streptococcus anginosus S. intermedius, Streptococcus MG and serological groups G ('minute' colony, type I) and F (type I). Most of the test strains contain the peptidoglycan type Lys-Ala_1–3. Only streptococci of serogroups R and S reveal a directly cross-linked peptidoglycan. Rhamnose was found as characteristic component of all cell wall polysaccharides. The impact of our results on the systematics of classical pyogenic streptococci will be discussed.     

Nuclear ribosomal DNA (nrDNA) concerted evolution in natural and artificial hybrids of Armeria (Plumbaginaceae)

Nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences from artificial hybrids and backcrosses between Armeria villosa ssp. longiaristata and A. colorata were studied to assess the possible effects of concerted evolution in natural hybrids. F1 artificial hybrids show the expected pattern of additive polymorphisms for five of the six variable sites as estimated from direct sequences. However, homogenization of polymorphism is already observed in the F2, and is biased towards A. colorata except for one site. In backcrosses, an expected tendency towards homogenization of polymorphic sites in the direction of the recurrent parent is observed for five sites, although this does not necessarily imply concerted evolution. Conversely, the sixth site appears to elude such a mechanism and thus provides additional support for the occurrence of biased concerted evolution. Our findings are relevant to interpreting phylogeographic patterns involving gene flow and are also consistent with the hypothesis of a hybrid origin of A. villosa ssp. carratracensis.     

Changes in the rRNA levels of specific microbial groups in activated sludge during sample handling and storage

AIMS: To quantitatively analyse the changes in group-specific rRNA levels in activated sludge as a function of sample handling and storage procedure. METHODS AND RESULTS: Quantitative membrane hybridizations with (32)P-labelled oligonucleotide probes were used to analyse the effects of different sample handling and storage conditions on the relative rRNA levels of the alpha, beta, and gamma-Proteobacteria, the Cytophaga-Flavobacteria group, and the mycolic acid-containing actinomycetes in activated sludge. Group-specific rRNA levels, expressed as percentages of total 16S rRNA detected with a universal probe, in samples maintained at room temperature significantly changed after 48 h. Group-specific rRNA levels in samples treated with chloramphenicol showed significant change after 72 h. CONCLUSIONS: Sample storage at room temperature is a viable option if freezing or analysis can be performed within 24 h, while treatment with chlorampenicol can extend that time to at least 48 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Handling, shipping, and storage of environmental samples under several conditions may result in inaccurate determination of the microbial populations in microbial ecology studies.     

Indexes to the reassociation and stability of solution DNA hybrids

Summary The computation, assumptions, and properties of DNA-hybrid stability and reassociation indexes were reviewed. Different methods of computing the same index typically yielded similar values. However, because dissociation curves change from asymmetric to symmetric as increasingly divergent DNAs are compared, adequate determination of mode required fitting a complex function. Delta Tm, delta mode, and delta T50H correlated well up to ca. 12, and all were found to be useful indexes of genomic similarity in that range. They also exhibited similar levels of error, even though T50H comprises a percent reassociation component with relatively large variance. At greater distances, the delta Tm scale became markedly compressed because of the boundary imposed by the temperature of hybrid formation (incubation temperature). Though not compressed or technically limited by it, delta mode and delta T50H could not be extrapolated with certainty below the incubation temperature. Among theoretical problems discussed: Tm and mode index an increasingly small percentage of the genome as the extent of reassociation decreases, and they may compare different genomic segments as DNAs become highly diverged. T50H relies upon the assumptions that all sequences evolve at a constant rate and that reassociation behavior is the same among all sequences regardless of their extent of divergence. Tm and T50H may be biased by selfhybridization of repetitive elements or cross-hybridization of paralogous sequences. Delta mode is free of such biases as long as the genomes under comparison are not too diverged. No index was found to be best in all circumstances.     

A hybridization and enzyme blocking method to enrich minor cDNA sequences

A method is presented which allows for the enrichment of low frequency cDNA sequences. The crucial step in the procedure is the hybridization of a pool of cDNA to homologous or heterologous RNA to a Rot value which will leave minor sequences in a single strand cDNA form while the major sequences form cDNA:RNA hybrids. This allows subsequent enzymatic differentiation between major and minor sequences resulting ultimately in the degradation of the major sequences. The procedure is general and simple as it requires no column chromatography step. The method is designed to integrate into a widely used cDNA cloning protocol and results either in double-stranded cDNA which can be used for molecular cloning or as a source of probes for hybridization.     

原位杂交技术及其在果树研究中的应用

原位杂交技术是近年来快速发展起来的一门新技术,本文介绍了原位杂交技术的基本原理、方法及其发展前景,以及该技术与其它生物学技术相结合而形成的一些新技术。综述了这些技术在果树研究中的应用情况。     

Genetic and ecological differentiation in the hybridizing campions Silene dioica and S. latifolia

Ecological differentiation is a major contributor to the generation and maintenance of biological diversity. We investigated habitat differentiation between and within sites in the fully cross-fertile and hybridizing Silene dioica and S. latifolia using amplified fragment length polymorphisms (AFLP) profiles and corresponding vegetation relevés around individual plants. Nineteen study sites in the Swiss Alps included pure sites and contact sites (both taxa present within 30 m). In pure sites and at contact sites, the two taxa showed consistently differentiated AFLP banding patterns across regions but few discriminating bands. This indicates that although the two taxa are weakly differentiated, current introgression has not led to genome-wide admixture. Only three putative early generation hybrids were detected at contact sites. The habitats of the two taxa differed between pure sites with S. dioica occurring in moister, colder, and less-disturbed sites than S. latifolia. However, asymmetric habitat overlap was evident within contact sites found in intermediate conditions that were more similar to S. latifolia sites. This situation might favor introgression from S. dioica into S. latifolia. Evidence for habitat-genotype associations within contact sites was weak making habitat-mediated selection against intermediate phenotypes of hybrids unlikely in the contact sites investigated. We suggest that other reproductive barriers together with dispersal limitation contribute to the rarity of early generation hybrids.     

Sexual and somatic hybridization in the genusLycopersicon

In recent years, a large number of reports have been published on the recovery of somatic hybrids in the genusLycopersicon and their potential use as a tool in plant breeding programs. Somatic hybridization as a way of enabling the incompatibility barriers which exist within the genusLycopersicon to be bypassed has attracted great interest. WildLycopersicon species harbor numerous interesting agronomic characteristics, which could be transferred to tomato by somatic hybridization. In particular, the production of asymmetric hybrids is explored as an approach to obtain the transfer of only a part of the nuclear genome of wildLycopersicon species. Considerable information is available on the fate of chloroplasts and mitochondria in fusion products inLycopersicon, and unfortunately, cybridization (transfer of chloroplasts and/or mitochondria) seems often difficult to achieve.