A rapid and cost effective protocol for plant genomic DNA isolation using regenerated silica columns in combination with CTAB extraction

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.

Preparation and Characterization of Super-paramagnetic Nano-beads for DNA Isolation

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.

An Efficient Procedure for DNA Isolation and Profiling of the Hyper Variable MtDNA Sequences

Present study describes the results of an efficient protocol for the isolation of good quality DNA from human saliva. The protocol includes collection of saliva in sterile specimen tubes, followed by the cell lysis. After formation of cell lysate, proteins wereextracted by phenol chloroform treatment for purification of DNA. The purified DNA was precipitated by adding equal volume of isopropanol to the treated supernatant. After isolation DNA pellet was washed with 70% ethanol, air-dried and was suspended in 30 pL of double distilled water. Best quality of DNA was extracted from the saliva samples and the PCR product was amplified for hyper-variable regions （HVI＆ HV2） of the mitochondrial DNA. The genes were cleaned with GeneAll gel elution kit （Gel SV） （Cat. No. 102-10） and sequenced accordingly. The DNA isolation protocol presented here is recommended for the isolation, best quality and yield of DNA from the human saliva.

A Rapid Method for the Isolation of Small Amount DNA from Citrus Early Embryo

[Objective] The present study aimed to establish a rapid method for the isolation of small amount DNA from citrus.[Method] By using the improved CTBA method,the genomic DNA was extracted respectively from 20,10,5 and 2.5 mg hybrid embryos of citrus,and then the DNA quality was detected and followed by SSR verification.[Result] The method was very simple and rapid,which needed less materials.In addition,the isolated DNA showed good purity with the OD260/OD280 of 1.8-2.1,and could meet the requirement for PCR-based technology,such as SSR,etc..[Conclusion] The method could be used for rapid extraction of small amount of genomic DNA from citrus.

Cloning of Banana Bunchy Top Virus Chinese Zhangzhou Isolate DNA 4 and the Promoter Activity of Its Non_coding Region

Banana bunchy top virus Chinese Zhangzhou isolate (BBTV-ZZ) DNA 4 was amplified by PCR and cloned. Sequence analysis showed that BBTV-ZZ DNA 4 is 1 039 nucleotides (nts) in length and this virus could be one member of BBTV Asian group. Transcriptional initiation site A, which is at the 269 nucleotide, was preliminarily determined by using 5' RACE method. BBTV-ZZ DNA 4 non-coding region was sub-cloned by PCR and inserted into upstream of gfp : : gus plant expression vector pCAMBIA 1304 to construct recombinant plasmid pTA2. Agrobacterium tumefaciens harboring pTA2 was injected into leaves of the tobacco (Nicotiana tabacum L. cv. Xanthi NC) via Agrobacterium-infiltration procedure. Transient expressions of GUS and GFP were determined in injected leaves 3 - 5 d later. GUS activities of pTA2, pCAMBIA 1304 injected and non-injected tobacco leaves respectively were 1.007 0 pmol MU(.)mug(-1.)min(-1), 2.069 0 pmol MU(.)mug(-1.)min(-1) and 0.021 4 pmol MU(.)mug(-1.)min(-1). Indirect ELISA for GFP in 1 mg total protein from pTA2, pCAMBIA 1304 injected and non-injected leaves showed an A(490 nm) value of 89.577, 100.440 and 3.287, respectively. These results showed that the non-coding region of BBTV-ZZ DNA 4 has a promoter activity not only in the virus replication in monocot, but also in driving the expression of a foreign gene in dicot plants.

Iron-Mediated Oxidative DNA Damage Detected by Fluorometric Analysis of DNA Unwinding in Isolated Rat Liver Nuclei

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.

Recycling Isolation of Plant DNA,A Novel Method

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.

Functional Metagenomics from the Rumen Environment—A Review

The rumen microbiome plays an essential role in ruminant physiology, nutrition and pathology as well as host immunity. A better understanding of rumen 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. There 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 Escherichia coli (E. coli). 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.

Effective Isolation of Retrotransposons and Repetitive DNA Families from the Wheat Genome

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.

Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Good quality deoxyribonucleic acid （DNA） is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction （PCR） applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyl trimethylammonium bromide （CTAB） method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone （PVP） and 13-mercaptoethanol were employed to man＇age phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 pg of good quality DNA, free of protein and RNA. The problems of DNA degradation,contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method, The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat （SSR） primers, This modified protocol can also be used to extract DNA from other woody plants having similar problems.