BACKGROUNDMitochondrial DNA mutations have been found in sensorineural deafness. The aim of this study was to compare three methods for extraction of nucleic acid from membranate inner ear tissue of rats.

METHODSAlkaline denaturation, a conventional phenol-chloroform method and Trizol reagent were respectively used to extract the slight nucleic acid from membranate inner ear tissue of rats. We assessed the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR).

RESULTSThe yield and purity (OD260/OD280) of DNA from inner ear tissue using the phenol-chloroform method was the highest of the three methods. Mitochondrial DNA (mtDNA) fragment can be amplified by PCR from nucleic acid prepared by all methods, while no nuclear DNA (nDNA) fragment can be amplified by method of alkaline denaturation. Both nuclear and mitochondrial genes could be amplified by reverse transcriptional PCR from the RNA prepared by Trizol reagent.

CONCLUSIONAdequate amount and high-quality of mtDNA, nDNA and RNA were obtained from unilateral membranate inner ear tissue of rats. Method of alkaline denaturation could be chosen when mtDNA without nDNA was needed, while phenol-chloroform method was suitable for extracting total DNA (including nDNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.

Animals ; DNA ; isolation & purification ; DNA, Mitochondrial ; isolation & purification ; Ear, Inner ; chemistry ; Polymerase Chain Reaction ; RNA ; isolation & purification ; Rats ; Rats, Wistar

A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.
Animals ; DNA, Complementary ; chemistry ; isolation & purification ; Gene Library ; RNA ; isolation & purification ; Sea Anemones ; genetics

Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Cetrimonium Compounds ; chemistry ; Chemical Precipitation ; DNA ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; isolation & purification ; Escherichia coli ; chemistry ; genetics ; Plasmids ; genetics ; isolation & purification

Quantification of residual DNA in animal-derived biological scaffold materials is one of technical specifications for evaluating decellularization process and immunotoxicity risk. Up to now, there have been no standard methods available for quantification of residues DNA in animal-derived biological scaffold materials. In this study, a three-step method, including proteinase K digestion, DNA purification and determination of DNA using fluorescence assay, was designed for residual DNA quantification. A parallel recovery experiment of standard DNA using the same protocol to test article determination was used for adjusting final results of residuul DNA amount. DNA purification based on magnetic beads enabled the experiments to get high accuracy and repeatability. The validation experiment showed that the three-step method had high sensitivity up to 6.25ng of DNA per sample with good linearity (recovery curve R2 > 0.99) in the concentration range of 3. 125-100ng, and 25-400ng per sample. This method is useful for determining micro or trace amount DNA remained in the biomaterials.
Animals ; Biocompatible Materials ; chemistry ; DNA ; analysis ; isolation & purification ; Materials Testing ; methods ; Tissue Scaffolds ; chemistry

AIMThere are many factors affecting hybridization in cDNA chip: DNA concentration immobilized on glass surface, spotting solution dissolving DNA, the concentration and purity of fluorescence-labeled probe, hybridization solution, hybridization temperature, hybridization time, Cy3- and Cy5-labeled probes, etc. In order to improve hybridization efficiency, tests were designed to optimize these factors.

METHODSFactors are changed one by one. Optimal values are selected by comparing those of different groups.

RESULTS AND CONCLUSIONThe efficient hybridization condition is as follows: 0.50 microgram.microL-1 DNA fragments resolved in 1 x Micro Spotting Solution (ArrayIt) hybridize with purified fluorescent probe in 0.5% SDS/10 x SSC at 42 degrees C.

Brain Chemistry ; DNA, Complementary ; chemistry ; Humans ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods ; RNA ; chemistry ; isolation & purification

OBJECTIVE: Comparing the differences in purity and yield among six methods of extracting human genomic DNA from whole blood, which included Classic Phenol-chloroform extraction, modified combined technique composed of improved Phenol-chloroform extraction and Chelex-100 extraction, Chelex-100 extraction, IQ, Qiagen and SP. METHODS: Ten samples of intravenous whole blood (5 mL/sample) were collected and human genomic DNA was extracted with these six methods. The purity and concentration of the DNA products were detected by ultraviolet spectrophotometry and fluorescent quantitation technique, the yield was calculated and tested with statistical software. RESULTS: The Chelex-100 extraction was inferior in DNA purity to other methods while the other five methods showed no statistical difference. Modified combined technique was the poorest and IQ was the best in yield among the six methods of extraction. Statistical result showed that the extraction with high quality kits was better than that with classic Phenol-chloroform extraction, Chelex-100 extraction and modified combined technique composed of improved Phenol-chloroform. There was statistical difference between them. CONCLUSION Comparing to Phenol-chloroform extraction and Chelex-100 extraction, high quality kits are more useful in DNA extraction from forensic materials.
Chloroform/chemistry* ; DNA/isolation & purification* ; Forensic Medicine/methods* ; Genomics/methods* ; Humans ; Phenol/chemistry* ; Reagent Kits, Diagnostic ; Resins, Synthetic/chemistry*

OBJECTIVE: To establish a new method for RNA and DNA co-extraction from the same sample by TRIzol reagent. METHODS: After the aqueous phase which contained total RNA was removed by traditional TRIzol method, the values of pH of the interphase phase and organic phase were adjusted. The DNA was precipitated with ethanol and purified with DNA IQ system. The purified DNA was measured in quality and quantity. As the template, it was amplified and typed by PCR-STR. The data was compared with that extracted by traditional TRIzol method. RESULTS: The DNA extracted by this modified method showed a better result of quality and quantity than that by traditional TRIzol method and a good STR typing. CONCLUSION The modified TRIzol method is advisable and reliable to simultaneously extract both DNA and RNA from the same sample. It could be used for individual identification and paternity testing to satisfy the need of forensic science.
Blood Chemical Analysis/methods* ; DNA/isolation & purification* ; DNA Fingerprinting ; Forensic Medicine ; Guanidines/chemistry* ; Humans ; Hydrogen-Ion Concentration ; Phenols/chemistry* ; Polymerase Chain Reaction ; RNA/isolation & purification* ; Reagent Kits, Diagnostic

A new flavonone, named as (2R, 3S)-pinobanksin-3-cinnamate(1), together with six known compounds, pinocem-brin (2), pinobanksin (3), 3-O-acetylpinobanksin (4), galangin (5), kumatakenin(6), and 3-methylkaempferol (7), were isolated from a 95% ethanol extract of seeds of Alpinia katsumadai through a combination of various chromatographic techniques, including silica gel and Sephadex LH-20. The structure of compound 1 was elucidated by spectroscopic data analysis. Compound 1 exhibits a potent neuroprotective effect against the corticosterone-damaged PC12 cells, which may be underlying the effect by scavenging intracellular ROS.
Alpinia ; chemistry ; Animals ; Cell Death ; drug effects ; Cholestenones ; chemistry ; isolation & purification ; pharmacology ; Cinnamates ; chemistry ; isolation & purification ; pharmacology ; DNA Fragmentation ; drug effects ; Neuroprotective Agents ; chemistry ; isolation & purification ; pharmacology ; Oxidative Stress ; drug effects ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; Seeds ; chemistry

Therapeutic monoclonal antibodies become the major product class within the biopharmaceutical market. Protein A as the first capture step is still dominant in current platforms for purification of monoclonal antibodies. In this study, we developed a new antibody harvest process that incorporates acidic treatment of cell harvest, demonstrating high process yield, improved clearance of host cell associated contaminants, like non-histone host cell protein, histone, DNA and heteroaggregates. Host protein contamination was reduced about 10-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Turbidity increase of eluted IgG upon pH neutralization was nearly eliminated. Residual levels of impurities in the protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. The mechanism of host cell associated contaminants removal during acidic treatment was also explored. After a polishing step by Capto adhere, host cell protein was reduced to less than 5 ppm, DNA less than 1 ppb, histone to undetectable level, heteroaggregates less than 0.01% with total IgG recovery around 87%. This efficient process can be easily integrated into current IgG purification platforms, and may overcome downstream processing challenges.
Antibodies, Monoclonal ; isolation & purification ; Biotechnology ; Chromatography, Affinity ; DNA ; Histones ; Hydrogen-Ion Concentration ; Immunoglobulin G ; isolation & purification ; Staphylococcal Protein A ; chemistry

We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3' end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5' end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a "sandwich" format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.
Colorimetry ; methods ; DNA Probes ; chemistry ; genetics ; DNA, Bacterial ; genetics ; Gold ; chemistry ; Humans ; Metal Nanoparticles ; chemistry ; Mycobacterium tuberculosis ; isolation & purification ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods