OBJECTIVE: To compare the concentration of teeth DNA extracted by three different pretreatment methods and to explore a simple, economical and practical pretreatment method with high concentration of extracted DNA from teeth. METHODS: A total number of 21 molars were collected from 7 corpses. The pretreatment of 3 molars from each individual was randomly performed by tooth crumb method, ball-milling method and liquid nitrogen milling method and 50 mg tooth crumb was weight and DNA was extracted by AutoMate Express forensic DNA extraction system. Subsequently, the concentration of DNA and corresponding STR genotyping of three methods were compared. RESULTS: The DNA concentration extracted by tooth crumb method, ball-milling method and liquid nitrogen milling method was 0.055 6-1.989 1 ng/μL, 0.036 6-1.175 6 ng/μL and 0.037 8-1.249 0 ng/μL, respectively. The DNA concentration obtained by tooth crumb method was higher (P < 0.05) and the success rate of STR genotyping was high. CONCLUSION Combined with AutoMate Express forensic DNA extraction system, tooth crumb method is an efficient and feasible method to extract DNA from teeth, which can be applied in forensic practice.
DNA/isolation & purification* ; DNA Fingerprinting/methods* ; Genotype ; Humans ; Tooth

DNA, Circular ; isolation & purification ; DNA, Viral ; isolation & purification ; Hepatitis B ; diagnosis ; Hepatitis B virus ; genetics ; Humans

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

OBJECTIVETo develop a rapid and effective method for genomic DNA extraction with magnetic bead-based semi-automatic system.

METHODSDNA was extracted from whole blood samples semi-automatically with nucleic acid automatic extraction system.The concentration and purity of samples was determined by UV-spectrophotometer. Orthogonal design was used to analyze the main effect of lysis time, blood volume, magnetic bead quantity and ethanol concentration on the DNA yield; also the 2-way interaction of these factors.

RESULTSLysis time, blood volume, magnetic bead quantity and ethanol concentration were associated with DNA yield (P<0.05), but no interaction existed. DNA yield was higher under the condition with 15 min of lysis time, 100 μl of blood volume, 80 μl of magnetic beads and 80 % of ethanol. A significant association was found between the magnetic bead quantity and DNA purity OD260/OD280 (P=0.008). Interaction of blood volume and lysis time also existed (P=0.013). DNA purity was better when the extracting condition was 40 μl of magnetic beads, 15 min of lysis time and 100 μl of blood volume. Magnetic beads and ethanol concentration were associated with DNA purity OD260/OD230 (P=0.017 and P<0.05), the result was better when magnetic beads was 40 μl and ethanol concentration was 80 %.

CONCLUSIONThe results indicate that the optimized conditions with 40 μl magnetic beads will generate higher quality of genomic DNA from the whole blood samples.

Analysis of Variance ; DNA ; blood ; isolation & purification ; Humans ; Immunomagnetic Separation ; methods

Brucella ; genetics ; isolation & purification ; DNA, Bacterial ; blood ; isolation & purification ; Polymerase Chain Reaction ; methods

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

Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.
Anions ; Chromatography, Ion Exchange ; methods ; Cryogels ; chemical synthesis ; DNA ; isolation & purification ; Genetic Vectors ; isolation & purification ; Plasmids ; isolation & purification ; Porosity

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

OBJECTIVETo develop a rapid and definite diagnostic test of bacterial enteritis caused by pathogenic enterobacteria, the most frequent etiologic agent of infectious enteritis in the world.

METHODSA set of conventional PCR assays were applied to detect and identify salmonella, shigella, and E. coli O157:H7 directly from pure culture and fecal samples. The general primers of pathogenic enterobacteria were located on the uidA gene, which were found not only in E. coli nuclear acid, but also in shigella and salmonella genes. Shigella primer was from ipaH gene whose coded invasive plasmid relative antigen existed both in plasmid and in genome. The primers of salmonella were designed from the 16SrRNA sequence. The primer of E. coli O157:H7 was taken from eaeA gene. Five random primers were selected for RAPD. The detection system included common PCR, semi-nested PCR and RAPD.

RESULTSThis method was more sensitive, specific and efficient and its processing was rapid and simple. For example, the method could be used to specifically detect and identify salmonella, shigella, and E. coli O157:H7, and its sensitivity ranged from 3 to 50 CFU, and its detection time was 4 hours.

CONCLUSIONThis PCR method, therefore, can serve as a routine and practical protocol for detecting and identifying pathogenic microorganisms from clinical samples.

DNA Primers ; DNA, Bacterial ; analysis ; Escherichia coli O157 ; isolation & purification ; Feces ; microbiology ; Humans ; Polymerase Chain Reaction ; Salmonella typhi ; isolation & purification ; Sensitivity and Specificity ; Shigella flexneri ; isolation & purification

7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct. Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg). However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified. In this study, we attempted to purify and identify the enzyme. Through several purification procedures, we obtained two proteins (32 kD and 29 kD). The larger protein co-migrated with Fpg in 12% SDS-PAGE gel. Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps. These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg.
Chromatography, Affinity ; DNA Damage ; DNA Repair ; Escherichia coli/enzymology* ; Nucleosidases/isolation & purification* ; Sequence Analysis, Protein