Use of non-radioactive digoxigenin-labeled DNA probes for RFLP analysis in rice

A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.     

A protocol for high-throughput extraction of DNA from rice leaves

Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less than 6 mo of storage at 4°C.     

水稻两种细胞质雄性不育“三系”及其F1杂交种线粒体DNA的RFLP分析

对水稻BT型和WA型细胞质的雄性不育系,相应保持系和恢复系以及杂种的mtDNA用12个线粒体探针进行了RFLP分析,结果如下(1)BT型和WA型不育系的mtDNA在组织结构上存在差异;(2)不育系的mtDNA与其保持系间存在显著差异,推测mtDNA与水稻的cms有关;(3)atp9探针检测到WA型不育系与F1之间的多态性,Frag36探针检测到BT型不育系与F1之间的多态性,Frag9探针检测到WA型和BT型不育系与其F1之间的多态性,证明核恢复基因影响mtDNA的结构;(4)对mtDNA的结构变异与细胞质雄性不育的关系进行了分析与探讨.     

Detection and differentiation of African coconut phytoplasmas: RFLP analysis of PCR-amplified 16S rDNA and DNA hybridisation

Phytoplasmas associated with lethal decline diseases of the coconut palm (Cocos nucifera) in west and east Africa were detected by the specific amplification of their 16S rRNA genes. The primers used were based on conserved mollicute-specific and coconut-phytoplasma 16S rRNA gene sequences. Phytoplasma 16S rDNA was amplified from all African decline affected palms, some periwinkle maintained phytoplasmas, but not from healthy palms, infected palms from Florida or from the Cocos spiroplasma and Acholeplasma sp. mollicutes. African phytoplasmas were also detected by DNA hybridisation using two probes from the palm lethal yellowing phytoplasma from Florida. Probes hybridised at moderate stringency to dot blots of lethal decline affected palms from Africa, indicating possible genetic relationships between different coconut phytoplasmas. RFLP analysis of rDNA fragments (length c. 1.45 kbp) detected polymorphisms, indicating that the pathogens found in west and east Africa are not identical. This provides a useful tool for further epidemiological studies of African coconut phytoplasmal diseases.     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Detection of citrus yellow mosaic virus by PCR and nucleic acid spot hybridisation using non-radioactive probes in commercial citrus species

Citrus yellow mosaic virus (CYMV) was detected by polymerase chain reaction (PCR) in leaf samples of sweet orange, Rangpurlime, Pumello pink and acid lime and also in twig bark, fruit rind, fruit juice except fruit rag of sweet orange cv. Sathgudi, where all the positive samples showed bright amplification of the 726 bp band except in fruit rind. The CYMV could be detected by the biotin labelled probe of CYMV up to 1:100 dilutions by TE and TNE extraction methods in sweet orange cv. Sathgudi as well as Rangpurlime.     

Identification of wheat-Agropyron cristatum monosomic addition lines by RFLP analysis using a set of assigned wheat DNA probes

A non-radioactive digoxigenin-labelled DNA method was used successfully to identify RFLP markers in 54 Triticum aestivum cv Chinese Spring — Agropyron cristatum (2n=28, genome PPPP) P-genome monosomic addition lines. Southern analysis using a set of 14 DNA probes identifying each homoeologous chromosome arm, combined with two restriction enzymes HindIII and EcoRI, indicated that six A. cristatum chromosomes (1P, 2P, 3P, 4P, 5P and 6P) and five A. cristatum chromosome arms (2PS, 2PL, 5PL, 6PS and 6PL) have been individually added to the wheat genome. The added chromosomes of three lines were Agropyron translocated chromosomes. It was also found that two addition plants possessed an Agropyron-wheat translocation. These results showed that RFLP analysis using the set of assigned wheat probes was a powerful tool in detecting and establishing homoeology of alien A. cristatum chromosomes, or arms, added to wheat, as well as in screening the alien addition material. The creation of the monosomic addition lines should be useful for the transfer of disease-resistance genes from A. cristatum to wheat.     

番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用

以番茄(Solanum lycopersicum L.cv.Micro-Tom)叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL(反应总体积为12.5μL),发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。     

Analysis of the mitochondrial DNA of the somatic hybrids of Solanum brevidens and S. tuberosum using non-radioactive digoxigenin-labelled DNA probes

Summary Mitochondrial (mt) DNAs of somatic hybrids obtained by electrical and chemical fusion of mesophyll protoplasts of S. brevidens and a dihaploid line of S. tuberosum PDH 40 were analysed by Southern hybridization using the digoxigenin-labelled mtDNA sequences nad5 or orf25. In the Southern analysis of the hybrid mtDNA probed with nad5, most of the 19 hybrids analyzed had an RFLP pattern similar, but not identical, to one of the parents, S. tuberosum, PDH40. Nineteen percent of the hybrids had most of the S. brevidens fragments. Five of the hybrids had an identical RFLP pattern to either one of the parents while another two hybrids had novel RFLP patterns. Similar results were obtained by Southern analysis with orf25. These results clearly show that mtDNA rearrangements had occurred at a high frequency in the somatic hybrids. There were no differences in the frequencies of rearrangements observed between the hybrids regenerated from chemical and electrical fusions.     

Consensus multiplex PCR–restriction fragment length polymorphism (RFLP) for rapid detection of plant mitochondrial DNA polymorphism

A fast, simple, and efficient approach, termed consensus multiplex PCR–RFLP, was developed and employed to detect mitochondrial (mt)DNA variation in three orchid species, Spiranthes hongkongensis, S. sinensis , and S. spiralis . Using multiplex PCR, three pairs of consensus mitchondrial primers were added simultaneously into each reaction tube to amplify three nonoverlapping introns located in the NADH dehydrogenase genes. Fragment length differences in the multiplex PCR amplicons were directly detectable between S. spiralis and the other two species. Further restriction analysis of the multiplex PCR amplicons revealed sufficient mtDNA polymorphism, suitable for phylogenetic studies at the interspecific level. This approach is well suited for large-scale population surveys of mitochondrial genome diversity in plants. Additionally, the maternal mode of inheritance of organelle genomes renders this approach valuable for rapid identification of the origin and specific parentage of hybrid or allopolyploid species.     

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

Linkage map of phenotype and RFLP markers in rice

The results from linkage mapping activities at Kyushu University during the last 10 years are summarized in this paper. The present paper concisely reveals present situation on linkage map of phenotype markers, the integration linkage map of phenotype and RFLP markers and the genetic stocks available. Some of the problems in this field, in addition, are pointed out and discussed.     

用PCR及RFLP方法分析支原体与植原体的同源性

将猪鼻支原体和泡桐丛枝病植原体的16S rDNA进行PCR扩增,分别得到一条1 kb左右的扩增片段。PCR扩增产物用限制性内切酶EcoRⅠ、HindⅢ、BamHⅠ、SalⅠ和SmaⅠ进行RFLP(限制性片段长度多态性)分析,发现用RFLP分析猪鼻支原体和泡桐丛枝病植原体16S rDNA序列同源性的相关系数为0.72。     

Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species

Abstract Dioxigenin-labelled synthetic DNA probes directed against the 16S rRNA were used for the direct detection of the periodontopathogenic bacteria Prevotella intermedia and Porphyromonas gingivalis in subgingival plaque by applying a DNA-RNA dot-blot hybridization procedure. The test was evaluated with 134 plaque samples from 26 patients with adult periodontitis or rapidly progressive periodontitis. The lower limit of detection was 10⁴–10⁵ bacteria/specimen. A semiquantitative assessment of the two species in each sample and in the corresponding periodontal site was achieved by this technique. It is possible to examine 80–90 samples within two days with low material costs.     

PCR preparation of DNA inserts from lambda and plasmid vectors for RFLP mapping

A simplified method is described for preparing insert DNA for labelling reactions to be used in Southern hybridization. This method works with sequences cloned into both plasmid and lambda phage, and eliminates many of the steps leading to the labelling reaction. Small quantities of hostE. coli or lambda phage carrying a probe sequence are lysed and amplified via the polymerase chain reaction using standard sequencing primers. Unincorporated nucleotides are removed by ethanol precipitation or gel purification and insert DNA is ready for radio-labelling. This method reduces the time and expense associated with conventional insert preparation, and greatly simplifies the use of sequences cloned into lambda phage.     

Discrimination of West Nile virus and Japanese encephalitis virus strains using RT-PCR RFLP analysis

West Nile (WN) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. Since an outbreak of WN encephalitis in humans and horses occurred in New York City in late August 1999, the possibility exists that WN virus will invade regions that have close links with the United States, such as Japan. We developed a genetic diagnostic method that discriminates between strains of WN virus and Japanese encephalitis (JE) virus. The method involves RT-PCR restriction fragment length polymorphism (RFLP) analysis with a RT-PCR primer set, a nested PCR primer set, and a restriction enzyme. We detected WN and JE viruses in experimentally infected animal brain, spleen, and serum samples. Our method is useful in distinguishing WN viruses from the endemic background of JE viruses, and in discriminating the highly virulent WN strain, which was isolated in New York in 1999, from other WN virus strains.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.     

Cloning and characterization of pejerrey mitochondrial DNA and its application for RFLP analysis

Probes were cloned, characterized, and developed for all regions of the mitochondrial DNA (mtDNA) of pejerrey Odontesthes bonariensis to provide the basis for the study of genetic diversity of South American atherinopsinii and to enable species identification from small amounts of tissue. The mtDNA was extracted from liver and cleaved with Eco RI, producing four fragments (7.4, 3.4, 3.1 and 2.9 kb) which were cloned using pUC118 plasmid vectors. Sequence analysis from both ends of the fragments showed that they encode tRNA (Asp, Phe, and Ser-TGA), 12 S rRNA, cytochrome oxidase (CO) II, NADH 4, 5, and 6, and the D-loop, and that the relative positions of these genes are identical to those in the mtDNA of other teleosts. A comparison of homology with carp mtDNA nucleotide sequences revealed that tRNA (Phe and Ser-TGA) and CO II were relatively conserved, whereas the D-loop region was highly divergent. The cloned mtDNA probes detected mtDNA fragments from about 800 ng of total DNA extracted from liver, muscle, and single embryos of O. bonariensis , and were effective for restriction length fragment polymorphism (RFLP) analysis of Patagonina hatcheri , the most distant atherinopsine relative of pejerrey. The cloned mtDNA probes may be useful for the analysis of genetic diversity and non-destructive species identification, including the examination of eggs, larvae and juveniles. The mtDNA sequences reported here provide the basis for the design of primers for PCR-based RFLP analysis.     

Comparative study on the chloroplast,mitochondrial and nuclear genome differentiation in two cultivated rice species,Oryza sativa and O. glaberrima,by RFLP analyses

Summary Restriction fragment length polymorphisms of chloroplast (ct), mitochondrial (mt) and nuclear DNA were investigated using eight cultivars of Oryza sativa and two cultivars of O. glaberrima. Relative variability in the nuclear and cytoplasmic genomes was estimated by a common measure, genetic distance. Based on the average genetic distances among ten cultivars for each genome, the evolutionary variabilities of the mitochondrial and nuclear genomes were found to be almost the same, whereas the variability of the chloroplast genome was less than half that of the other two genomes. Cluster analyses on ct and mt DNA variations revealed that chloroplast and mitochondrial genomes were conservative within a taxon and that their differentiations were well-paralleled with respect to each other. For nuclear DNA variation, an array of different degrees of differentiation was observed in O. sativa, in contrast with little variation in O. glaberrima. As a whole, differentiation between O. sativa and O. glaberrima was clearly observed in all three genomes. In O. sativa, no notable difference was found between the cultivars Japonica and Javanica, whereas a large differentiation was noticed between Japonica (including Javanica) and Indica. In all three genomes, the average genetic distances within Indica were much larger than those within Japonica (including Javanica), and almost similar between Japonica (including Javanica) and Indica. These facts indicate that differentiation in O. sativa was due mainly to Indica.