杂交保护检测技术的应用

自杂交保护检测(Hybridization Protection Assay,HPA)技术首次被报道以来,在很多领域都得到了应用。本文综述了HPA技术的主要原理特性和应用的研究进展,包括微生物的鉴定检测、端粒和端粒酶检测、基因突变检测和基因多态性分析、mRNA转录水平监测和其他方面的应用,并对HPA技术在我国应用的局限和应用前景进行分析。     

Post-pollination hybridization barriers in <Emphasis Type="Italic">Nicotiana</Emphasis> section <Emphasis Type="Italic">Alatae</Emphasis>

Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse, but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species × short-pistil species crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil species × long-pistil species. Taken together, these barriers can generally be described by a ‘pistil-length mismatch’ rule; in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hybridization biosensor using 2-nitroacridone as electrochemical indicator for detection of short DNA species of Chronic Myelogenous Leukemia

A new acridone derivative 2-nitroacridone (NAD) was synthesized in this paper, and it was found that NAD had excellent electrochemical activity on the glassy carbon electrode (GCE) with a couple reversible redox peaks at 0.051 V and 0.103 V, respectively. Voltammetry was used to investigate the electrochemical behavior of NAD and the interaction between NAD and salmon sperm DNA. In pH 4.0 phosphate buffer solution, the binding ratio between NAD and salmon sperm DNA was calculated to be 2:1 and the binding constant was 3.19 × 10⁵ L/mol. A Chronic Myelogenous Leukemia (CML, Type b₃a₂) DNA biosensor was developed by immobilizing covalently single-stranded CML DNA fragments to a modified GCE. The surface hybridization of the immobilized single-stranded CML DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using NAD as a novel electrochemical indicator, with a detection limit of 6.7 × 10⁻⁹ M and a linear response range of 1.8 × 10⁻⁸ M to 9.1 × 10⁻⁸ M for CML DNA. Selective determination of complementary ssDNA was achieved using differential pulse voltammetry (DPV).     

Synthesis and geometry of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates

The synthesis of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates is presented. High resolution (1)H and (13)C NMR spectral data for all diastereoisomers and single-crystal X-ray diffraction analysis for methyl (methyl 3-azido-2,3-dideoxy-beta-D-arabino-hexopyranosid)uronate are reported. The planarity of the 4-OAc and 5-COOMe groups as well as the orientations of the aglycone and azide groups in the crystal lattice is discussed. The influence of the 5-COOMe group on the pyranose ring conformation is considered.     

Inheritance of leaf geranylflavanone production and seed production within and among chemically distinct populations of Mimulus aurantiacus

Most models to account for variation in defensive chemical production in plants assume that defensive chemical production is a quantitative trait determined by the additive effects of many genes, and that defensive chemical production is genetically negatively correlated with fitness. The inheritance of quantities of geranylflavanones and seed production, an estimate of female fitness, was studied in reciprocal crosses between several chemically distinct populations of Mimulus aurantiacus to test those assumptions. Genetic analyses using reciprocal crosses were used to test for maternal or paternal genetic effects. The quantities of individual geranylflavanones of the hybrids generally were inherited with dominance or over-dominance. Reciprocal genetic effects were rarely found. Within populations, leaf resin production in M. aurantiacus was independent of seed production. Results are consistent with previous reports suggesting that any evolutionary change in geranylflavanone production within populations may be constrained by low levels of genetic variation in geranylflavanone production.     

Ideal phenotypes and mismatching haplotypes – errors of mtDNA treeing in ants (Hymenoptera: Formicidae) detected by standardized morphometry

A total of 401 nest samples of Formica lugubris Zetterstedt, F. pratensis Retzius, F. aquilonia Yarrow, F. rufa Linnaeus, and F. polyctena Förster, covering the entire Palaearctic range of these species and including 2100 individual workers, was phenotypically investigated by a system of standardized morphometry, pairwise removal of allometric variance, and canonical discriminant functions. A mitochondrial DNA fragment including the cytochrome b gene was sequenced in 148 samples from basically the same range. In the more difficult F. pratensis vs. F. lugubris case, the phenotypic system correctly determined 99.6% of all nest samples, and 95.1% with p<0.05. In all other pairwise species discriminations any nest sample was correctly determined with p<0.01, and three samples with hybrids F. rufa×lugubris were identified. At four localities in the Pyrenees and the Urals, 9 samples with F. pratensis phenotypes (7 of them ideal) but F. lugubris mtDNA haplotypes could be identified, resulting in 14.5% of phenotype/haplotype mismatches. A local dominance of this mismatch combination was observed at one Pyrenean and one Ural locality. There was no indication of an F. pratensis haplotype associated with an F. lugubris phenotype. One ideal F. polyctena phenotype was associated with an F. aquilonia haplotype in a sample from the Urals, and one ideal F. aquilonia phenotype was combined with an F. lugubris haplotype in a sample from central Siberia, resulting in overall phenotype/haplotype mismatch frequencies of 12.5% and 11.1%, respectively. We conclude that all these samples cannot represent actual F₁ hybrids but are the result of hybridizations in the past followed by unidirectional purging of the nuclear genome. Whether this process of purging worked very fast or over longer periods of population history, and whether or not it was complete or incomplete, cannot be assessed from the available information. These facts of hybridizing in two thirds of the W Palaearctic wood ant species, of extreme regional hybrid frequencies (up to 26%), of unidirectional purging of nDNA associated with mismatching mtDNA haplotypes, and of occasional achievement of local dominance of these mismatch combinations, may serve as urgent warning not to perform isolated mtDNA phylogenetic studies without a geographically and locally wide sampling basis and without control by nDNA information or reliable phenotypic determination. The latter two systems definitely have superior significance when conflicts with mtDNA indications arise.     

Block copolymer-oligonucleotide conjugates for genotyping on microarrays

Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models.     

两个不同地区东方田鼠杂交子代RAPD标记分析

目的研究不同地区东方田鼠杂交后产下的子代的遗传特性.方法筛选4条10bp随机引物对宁夏和洞庭湖地区东方田鼠杂交子代的基因组进行随机扩增多态DNA(RAPD)分析,并对不同地区亲代以及子代相互之间的基因组DNA进行相似性分析.结果①所有东方田鼠均有相同的扩增片段出现;②两个不同地区的亲代分别有特异性片般;③亲代的DNA带型均能在子代中找到;④同一胎次东方田鼠之间基因共享度大约在72%～96%之间.结论RAPD分析能在一定程度上反映出东方田鼠种的特性以及亚种的特异性,而且同一胎次之间基因共享度较高.     

人载脂蛋白E7转基因小鼠肾脏cDNA削减文库与cDNA文库

目的　以人apoE7基因制备的高脂血症转基因小鼠模型已建成 ,要用于探查这一突变基因致病的分子机制 ,首先揭示人apoE7在小鼠体内诱发了那些基因的表达异常 ,是重要的环节之一。cDNA削减文库是寻找异常高表达的已知和未知基因有效的手段 ,而cDNA文库更是获取全长基因所必须。方法　自正常小鼠与转基因小鼠肾脏同时提取总RNA ,再分离mRNA ,逆转录制备cDNA后 ,用两次分子杂交削除转基因鼠cDNA中与正常小鼠相同的部分 ,再用PCR与择需PCR法扩增转基因鼠特异的基因 ,克隆入pGEM T载体后 ,制备削减文库。未经削减的转基因小鼠cDNA克隆入λgt11载体 ,制备基因表达文库。结果和结论　自高脂血症转基因小鼠肾脏构建的削减文库得到约 4 0 0个削减克隆 ,测定了部分克隆的序列 ,并进行同源性比较。λgt11 cDNA文库滴度 1 7× 10 7pfu ml,随机测定的克隆其插入片段长约 5 0 0bp以上。     

The extremely rapid oligonucleotide hybridization and high throughput detection of microbial gene sequences using fluorescence polarization

The hybridization of oligonucleotide sequences complementary to the genes of Shiga toxins (verotoxins) types 1 and 2 of enterohaemorrhagic Escherichia coli (EHEC) and human hepatitis C virus (HCV) was monitored using fluorescence polarization under the reaction condition of high salt concentration (0.8 M NaCl), which was optimized to obtain a higher rate of hybridization. The time courses of hybridization of fluorescently labeled oligomers (probe DNAs) with the amplified DNA or RNA of the genes were recorded. Two methods, the asymmetric PCR and NASBA, were used to amplify the genetic DNA of Shiga toxins and that of RNA in HCV, respectively. Probe DNA sequences were designed which hybridized extremely rapidly with amplicons of the genes of Shiga toxins types 1 and 2 and that of HCV. In the cases using the three different DNA probes, the hybridization was 90% complete in about 1 min, considerably faster than that of the 3 min reported previously. The rapidity of this hybridization could not be explained by the melting temperature or the G+C content of the probe sequences but its relationship with high order structure of the single stranded DNA or RNA of the amplicons in the solution was strongly suggested.