DNA Extraction

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-...

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of ...

In this article I am trying to find out if the temperature of Ethanol effects the amount of DNA extracted from Strawberries. I got this experiment idea when I went on a tour at the BC Cancer Agency. There, they told us what they did, and how they read and stored DNA.They also explained where they stored the information from DNA. We also got to extract DNA from a strawberry. Now thats where my first thought came from!

An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2h and 30min with <or=0.8bp allele typing accuracy. The minimal amount of DNA required for a complete DNA profile is 100 copies. To critically evaluate the capabilities of our portable microsystem as well as its compatibility with crime scene investigation processes, real-time STR analyses were carried out at a mock crime scene prepared by the Palm Beach County Sheriff's Office (PBSO). Blood stain sample collection, DNA extraction, and STR analyses on the portable microsystem were conducted in the field, and a successful "mock" CODIS hit was generated on the suspect's sample within 6h. This demonstration of on-site STR analysis establishes the feasibility of real-time DNA typing to identify the contributor of probative biological evidence at a crime scene and for real-time human identification.

Background: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60–80%.Aims: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction.Method: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step.Results: This method produced a 100%

The objective of this study was to estimate the Trypanosoma evansi infection rate and epizootical status of wild and domestic animals from the Brazilian Pantanal region using a standardized polymerase chain reaction (PCR). We used a simple DNA extraction method based on Chelex resin (BioRad, USA) on blood eluted from filter paper confetti. Primers directed to repetitive nuclear DNA sequences were used in the PCR, and could detect 30  fg of T. evansi DNA. The analytical specificity of the assay was evaluated using T. evansi, T. rangeli, T. cruzi, Leishmania braziliensis, Crithidia fasciculata and Herpetomonas muscarum DNAs as templates and the technique showed the expected 164 bp specific band solely for Trypanozoon trypanosomes. The application of the standardized PCR protocol in 274 field samples from domestic and wild mammals from the Rio Negro (Brazilian Pantanal region), showed a general infection rate of 10.2% while the traditional parasitological technique (direct search of the protozoan by the microematocrit centrifugue technique) was able to determine infection in only 1.1% of the animals. The peccaries and feral pigs were found to be the animals most frequently infected with T. evansi (24.4% and 30.7%, respectively). Both sampling and extraction methods used herein, showed to be simple and efficient to be applied in epidemiological surveys using PCR.

Most of the established molecular techniques in plant molecular biology rely on the reliability of extraction methods to obtain nucleic acids from a given environmental source. The development of protocols for isolating both RNA and DNA from the same sample is recently becoming a major concern especially when the sample is small. Despite a number of methods that describe the simultaneous isolation of RNA and DNA from the same sample have been reported, most of these methods have yet to be optimized to deal with the reduced quantity and compromised quality of samples encountered in plant genetics analysis. Neolamarckia cadamba (Roxb.) Bosser was chosen in the present study due to its commercial value and fast growing ability. Total RNA was successfully isolated from the leaf tissue with subsequent recovery of DNA from the extraction mixture through alcohol precipitation. Reverse transcriptase‐Polymerase Chain Reaction (RT‐PCR) was performed and arbitrary primers that produced reproducible, scorable and informative bands were selected for randomly amplified polymorphic DNA (RAPD) analysis. The amplicons of RT-PCR and RAPD were successfully obtained from all isolates, indicating that the extracted nucleic acids were intact and pure enough for quantitative molecular analysis.