Isolation of high quality DNA from multiple samples can be both time-consuming and expensive. We have developed a combined protocol to reduce the time component of the hexadecyltrimethylammonium bromide (CTAB) extra...Isolation of high quality DNA from multiple samples can be both time-consuming and expensive. We have developed a combined protocol to reduce the time component of the hexadecyltrimethylammonium bromide (CTAB) extraction method and reduced costs by regenerating the silica columns used to purify genomic DNA. We present data that shows, by in- creasing the temperature used during the CTAB method, the time required to extract crude genomic DNA can be reduced. We show that silica columns can be regenerated using HCI and still maintain their DNA-binding capacity. Furthermore, we show both spectrophotometrically, and by restriction enzyme cutting, that the quality of the eluted DNA is high. Critically, using both genomic DNA from pea and perennial ryegrass we demonstrate, using species-specific PCR primers, that there is no carry-over of DNA from repeated use of a single column. The main advantages of the method are high yield, high quality, cost effectiveness and time-saving. This method could satisfy demand when large numbers of plant genomic DNA samples are required, for example from targeting induced local lesions in genomes (TILLING) populations.展开更多
Unique coupling reagent, bis-(2-hydroxyethyl methacrylate) phosphate was used to prepare coated and functionalized superparamagnetic nanobeads, leading to a simple, effective method for coating the nanobeads. With th...Unique coupling reagent, bis-(2-hydroxyethyl methacrylate) phosphate was used to prepare coated and functionalized superparamagnetic nanobeads, leading to a simple, effective method for coating the nanobeads. With this method, the thickness of the coating layer and the functional group contents on the nano-beads could be controlled by changing the quantity of the coated monomers. The nanobeads were characterized by means of transmission electron microscopy (TEM) and Fourier transformation infrared spectroscopy (FTIR). The carboxyl-modified magnetic nano-beads were employed to streamline the protocol of isolation of genomic DNA from the human whole blood.展开更多
Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of f...Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of five antioxidants (catalase, superoxide dismutase, dimethyl sulfoxide, glutathione and diallyl sulfide) on this oxidative nuclear damage were also investigated. At the 0.05 level for statistical significance, iron induced concentration-dependent DNA degradation, and this effect was enhanced by ascorbate and bleomycin. The antioxidants catalase, dimethyl sulfoxide, and diallyl sulfide significantly reduced the iron-ascorbate-induced DNA damage, whereas superoxide dismutase and dimethyl sulfoxide significantly reduced iron-bleomycin-induced damage. Glutathione significantly increased the iron-bleomycin-induced DNA damage. These results suggest that the reactive oxygen species generated by iron, iron-ascorbate, and iron-bleomycin are responsible for the DNA strand breaks in isolated rat liver nuclei.展开更多
DNA is one of the most basic and essential genetic materials in the field of molecular biology. To date, isolation of sufficient and good- quality DNA is still a challenge for many plant species, though various DNA ex...DNA is one of the most basic and essential genetic materials in the field of molecular biology. To date, isolation of sufficient and good- quality DNA is still a challenge for many plant species, though various DNA extraction methods have been published. In the present paper, a recycling DNA extraction method was proposed. The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method. This modified CTAB method was tested in eight plant species, wheat, sorghum, barley, corn, rice, Brachy- podium distachyon, Miscanthus sinensis and tung tree. The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species. The DNA samples were good templates for PCR amplification of both ISSR and SSR markers. The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods, and thus is an effective and universal DNA isolation method.展开更多
<div style="text-align:justify;"> <span style="font-family:Verdana;">The rumen microbiome plays an essential role in ruminant physiology, nutrition and pathology as well as host immunit...<div style="text-align:justify;"> <span style="font-family:Verdana;">The rumen microbiome plays an essential role in ruminant physiology, nutrition and pathology as well as host immunity. A better understanding of rumen</span><span style="font-family:Verdana;"> microbial processes and identification of which populations are responsible for specific functions within the rumen microbiome will lead to better management and sustainable utilization of the available feed base while maintaining a low environmental impact. Recent advance in the culture independent method of microbiology such as metagenomics, unravels potentially the rumen microbial process. Th</span><span style="font-family:Verdana;">ere are two basic types of metagenomics studies: Sequence-based and function-based metagenomics. Sequence-based metagenomics involves sequencing and analysis of DNA from environmental samples. Its purpose is to assemble genomes, identify genes, find complete metabolic pathways, and compare organisms of different communities. Whereas functional metagenomics is the study of the collective genome of a microbial community by expressing it in a foreign host usually </span><i><span style="font-family:Verdana;">Escherichia</span></i> <i><span style="font-family:Verdana;">coli</span></i><span style="font-family:Verdana;"> (</span><i><span style="font-family:Verdana;">E.</span></i> <i><span style="font-family:Verdana;">coli</span></i><span style="font-family:Verdana;">)</span><i><span style="font-family:Verdana;">.</span></i><span style="font-family:Verdana;"> It is a promising approach unearthing novel enzymes even from yet to culture rumen microbiota. Further advances in the screening techniques promise vast opportunities to rumen microbiologists, and animal nutritionist. The identification of novel enzyme through functional metagenomics consists of three parts: rumen sample collection;DNA library construction and screening of individual clone. Functional metagenomics was successfully applied to identify different antibiotics, hydrolytic enzymes, antibiotic resistance genes, and many other functions;moreover, it allowed characterization of genes encoding enzymes with a particular activity, which represents completely novel sequence. There are a number of outputs from functionally screened rumen product such as carbohydrate active enzymes (CAZymes) that can break down plant cell walls. Company involved commercialization of metagenomics research such as Syngenta, Genencor International, BRAIN etc., has produced many biological molecules of commercial interest. The aim of this paper is to elucidate functional metagenomics, from rumen environment and its potential for commercial purpose.</span> </div>展开更多
New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cl...New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO1091, the recognition sites of which consisted of 5'-PuGGNCCPy-3' multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach, pTm6 showed identity with integrase domains of the barley Tyl-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.展开更多
基金funding and the PhD Scholarship for Fu Zeyu from the New Zealand Foundation for Arable Researchfunding for Song Jiancheng from the National Natural Science Foundation of China (31371616)
文摘Isolation of high quality DNA from multiple samples can be both time-consuming and expensive. We have developed a combined protocol to reduce the time component of the hexadecyltrimethylammonium bromide (CTAB) extraction method and reduced costs by regenerating the silica columns used to purify genomic DNA. We present data that shows, by in- creasing the temperature used during the CTAB method, the time required to extract crude genomic DNA can be reduced. We show that silica columns can be regenerated using HCI and still maintain their DNA-binding capacity. Furthermore, we show both spectrophotometrically, and by restriction enzyme cutting, that the quality of the eluted DNA is high. Critically, using both genomic DNA from pea and perennial ryegrass we demonstrate, using species-specific PCR primers, that there is no carry-over of DNA from repeated use of a single column. The main advantages of the method are high yield, high quality, cost effectiveness and time-saving. This method could satisfy demand when large numbers of plant genomic DNA samples are required, for example from targeting induced local lesions in genomes (TILLING) populations.
文摘Unique coupling reagent, bis-(2-hydroxyethyl methacrylate) phosphate was used to prepare coated and functionalized superparamagnetic nanobeads, leading to a simple, effective method for coating the nanobeads. With this method, the thickness of the coating layer and the functional group contents on the nano-beads could be controlled by changing the quantity of the coated monomers. The nanobeads were characterized by means of transmission electron microscopy (TEM) and Fourier transformation infrared spectroscopy (FTIR). The carboxyl-modified magnetic nano-beads were employed to streamline the protocol of isolation of genomic DNA from the human whole blood.
文摘Studies were performed to determine the extent of nuclear DNA degradation induced by iron, iron-ascorbate, or iron-bleomycin under aerobic conditions in a model system using isolated rat liver nuclei. The effects of five antioxidants (catalase, superoxide dismutase, dimethyl sulfoxide, glutathione and diallyl sulfide) on this oxidative nuclear damage were also investigated. At the 0.05 level for statistical significance, iron induced concentration-dependent DNA degradation, and this effect was enhanced by ascorbate and bleomycin. The antioxidants catalase, dimethyl sulfoxide, and diallyl sulfide significantly reduced the iron-ascorbate-induced DNA damage, whereas superoxide dismutase and dimethyl sulfoxide significantly reduced iron-bleomycin-induced damage. Glutathione significantly increased the iron-bleomycin-induced DNA damage. These results suggest that the reactive oxygen species generated by iron, iron-ascorbate, and iron-bleomycin are responsible for the DNA strand breaks in isolated rat liver nuclei.
基金supported by the National Natural Science Foundation of China(Grant Nos.31030055 and 30870233)the Important Directional Program of Knowledge Innovation Project of Chinese Academy of Sciences(Grant No. KSCX2-YW-Z-0722)
文摘DNA is one of the most basic and essential genetic materials in the field of molecular biology. To date, isolation of sufficient and good- quality DNA is still a challenge for many plant species, though various DNA extraction methods have been published. In the present paper, a recycling DNA extraction method was proposed. The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method. This modified CTAB method was tested in eight plant species, wheat, sorghum, barley, corn, rice, Brachy- podium distachyon, Miscanthus sinensis and tung tree. The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species. The DNA samples were good templates for PCR amplification of both ISSR and SSR markers. The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods, and thus is an effective and universal DNA isolation method.
文摘<div style="text-align:justify;"> <span style="font-family:Verdana;">The rumen microbiome plays an essential role in ruminant physiology, nutrition and pathology as well as host immunity. A better understanding of rumen</span><span style="font-family:Verdana;"> microbial processes and identification of which populations are responsible for specific functions within the rumen microbiome will lead to better management and sustainable utilization of the available feed base while maintaining a low environmental impact. Recent advance in the culture independent method of microbiology such as metagenomics, unravels potentially the rumen microbial process. Th</span><span style="font-family:Verdana;">ere are two basic types of metagenomics studies: Sequence-based and function-based metagenomics. Sequence-based metagenomics involves sequencing and analysis of DNA from environmental samples. Its purpose is to assemble genomes, identify genes, find complete metabolic pathways, and compare organisms of different communities. Whereas functional metagenomics is the study of the collective genome of a microbial community by expressing it in a foreign host usually </span><i><span style="font-family:Verdana;">Escherichia</span></i> <i><span style="font-family:Verdana;">coli</span></i><span style="font-family:Verdana;"> (</span><i><span style="font-family:Verdana;">E.</span></i> <i><span style="font-family:Verdana;">coli</span></i><span style="font-family:Verdana;">)</span><i><span style="font-family:Verdana;">.</span></i><span style="font-family:Verdana;"> It is a promising approach unearthing novel enzymes even from yet to culture rumen microbiota. Further advances in the screening techniques promise vast opportunities to rumen microbiologists, and animal nutritionist. The identification of novel enzyme through functional metagenomics consists of three parts: rumen sample collection;DNA library construction and screening of individual clone. Functional metagenomics was successfully applied to identify different antibiotics, hydrolytic enzymes, antibiotic resistance genes, and many other functions;moreover, it allowed characterization of genes encoding enzymes with a particular activity, which represents completely novel sequence. There are a number of outputs from functionally screened rumen product such as carbohydrate active enzymes (CAZymes) that can break down plant cell walls. Company involved commercialization of metagenomics research such as Syngenta, Genencor International, BRAIN etc., has produced many biological molecules of commercial interest. The aim of this paper is to elucidate functional metagenomics, from rumen environment and its potential for commercial purpose.</span> </div>
基金Supported by Grants-in-Aid for Scientific Research (01760004 and 04760006) from the Japanese Ministry of Education,Culture,Sports,Science and Technology (MEXT)
文摘New classes of repetitive DNA elements were effectively identified by isolating small fragments of the elements from the wheat genome. A wheat A genome library was constructed from Triticum monococcum by degenerate cleavage with EcoO1091, the recognition sites of which consisted of 5'-PuGGNCCPy-3' multi-sequences. Three novel repetitive sequences pTm6, pTm69 and pTm58 derived from the A genome were screened and tested for high copy number using a blotting approach, pTm6 showed identity with integrase domains of the barley Tyl-Copia-retrotransposon BARE-1 and pTm58 showed similarity to the barley Ty3-gypsy-like retrotransposon Romani. pTm69, however, constituted a tandem array with useful genomic specificities, but did not share any identity with known repetitive elements. This study also sought to isolate wheat D-genome-specific repetitive elements regardless of the level of methylation, by genomic subtraction. Total genomic DNA of Aegilops tauschii was cleaved into short fragments with a methylation-insensitive 4 bp cutter, Mbol, and then common DNA sequences between Ae. tauschii and Triticum turgidum were subtracted by annealing with excess T. turgidum genomic DNA. The D genome repetitive sequence pAt1 was isolated and used to identify an additional novel repetitive sequence family from wheat bacterial artificial chromosomes with a size range of 1 395-1 850 bp. The methods successfully led pathfinding of two unique repetitive families.