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DNA Recovery (dna + recovery)
Selected AbstractsOptimization of DNA Extraction from Low-Yield and Degraded Samples Using the BioRobot® EZ1 and BioRobot® M48JOURNAL OF FORENSIC SCIENCES, Issue 5 2006Ram Kishore Ph.D. ABSTRACT: Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots® EZ1 or BioRobots® M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot® EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1. [source] DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 2 2006Gian Marco Luna Summary In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H,) and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of ,-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples. [source] The Use of Hemastix® and the Subsequent Lack of DNA Recovery Using the Promega DNA IQTM SystemJOURNAL OF FORENSIC SCIENCES, Issue 6 2009Hiron Poon M.Sc. Abstract:, Following implementation of our automated process incorporating the Promega DNA IQTM system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix® reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix® chemistries on the DNA IQTM system. Our results indicate that one or more chemicals imbedded in the Hemastix® reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQTM magnetic bead technology. The 3,3,,5,5,-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix® chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix® chemistries on the DNA IQTM system forced us to adopt an indirect approach using filter paper to carry out the presumptive test. [source] Optimization of DNA Extraction from Low-Yield and Degraded Samples Using the BioRobot® EZ1 and BioRobot® M48JOURNAL OF FORENSIC SCIENCES, Issue 5 2006Ram Kishore Ph.D. ABSTRACT: Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots® EZ1 or BioRobots® M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot® EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1. [source] Liver-targeted doxorubicin: effects on rat regenerating hepatocytesLIVER INTERNATIONAL, Issue 3 2004Giuseppina Di Stefano Abstract: Background/Aims: The conjugate of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) has the potential of improving DOXO efficacy in the treatment of hepatocellular carcinomas (HCCs) expressing the asialoglycoprotein receptor (ASGP-R). In view of an adjuvant chemotherapy with L-HSA,DOXO after the surgical removal of the tumour, in the present experiments we verified whether DOXO accumulation produced by the conjugate can impair the liver regeneration following hepatic resection in non-cirrhotic liver. Methods: Using saline-injected hepatectomised rats as controls, we studied the effects of the conjugate on the ultrastructure of regenerating hepatocytes and evaluated [3H]thymidine incorporation, mitotic index and rate of DNA recovery in the liver remnant. Results: L-HSA,DOXO caused a selective drug accumulation in liver remnant, with low DOXO levels in extra-hepatic tissues. It did not change the ultrastructure of hepatocytes and did not increase serum alanine aminotransferase. It decreased [3H]thymidine incorporation and mitotic index, causing a moderate delay in hepatic DNA recovery. Conclusions: The experiments indicate a substantial resistance of rat regenerating hepatocytes to high intracellular concentrations of DOXO. They support the possibility of using L-HSA,DOXO in an adjuvant chemotherapy after the surgical removal of HCCs which maintain the ASGP-R. [source] Recovery of plant DNA using a reciprocating saw and silica-based columnsMOLECULAR ECOLOGY RESOURCES, Issue 1 2007PATRICK J. ALEXANDER Abstract The time needed for hand grinding and the cost of commercially available extraction kits remain to be the major limitations in plant DNA extraction for many researchers. We present inexpensive techniques for (i) simultaneously machine grinding large numbers of plant samples for DNA extraction using a commercially available reciprocating saw; and (ii) DNA recovery using silica column-based extractions similar to that used in some commercially available kits. Used together, these allow for the rapid recovery of plant DNA at relatively low cost. Furthermore, these methods appear to be widely applicable within plants with good yields recovered in test extractions across major plant groups (ferns, gymnosperms, monocots and eudicots). [source] Technical note: Efficiency of total demineralization and ion-exchange column for DNA extraction from boneAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 1 2010Seung Bum Seo Abstract We investigated whether a combination of recently introduced methods, total demineralization and ion-exchange columns, would increase DNA recovery from old bone. Ten bone samples taken after a burial period of ,60 years were used in this study. Bone powder was digested using total or incomplete demineralization. DNA was extracted by the standard organic method. The DNA extract was purified with ion-exchange columns or QIAquick® spin columns. The efficiency of different DNA extraction methods was compared in terms of DNA concentration, inhibitors generated by real-time PCR, and conventional STR typing results. The mean DNA concentration using the total demineralization method is ,3 times higher than that using the incomplete demineralization method. For DNA purification, the method using QIAquick® spin columns appeared to yield approximately double the DNA than the method using ion-exchange columns. Furthermore, 2 out of 10 samples showed higher levels of inhibition with CT values of IPC ,30 cycles when using only ion-exchange columns. In STR results, total demineralization yielded more locus profiles by 4.2 loci than incomplete demineralization, and QIAquick® spin columns also yielded more locus profiles by 3.5 loci than ion-exchange columns. Total demineralization of bone powder significantly increased DNA yield and improved STR typing results. However, the use of ion-exchange columns was not efficient when compared with the method using QIAquick® spin columns. It is suggested that the combination of total demineralization and QIAquick® spin columns lead to greatly improved STR typing results. Am J Phys Anthropol 2010. © 2009 Wiley-Liss, Inc. [source] A simple and efficient method for extraction of PCR-amplifiable DNA from chicken eggshellsANIMAL SCIENCE JOURNAL, Issue 2 2009Kazuhiro RIKIMARU ABSTRACT Recently, we reported a method for discriminating a Japanese brand of chicken, the Hinai-jidori. As an application of this method for discriminating Hinai-jidori eggs, we here report an efficient method for extracting maternal DNA from eggshells. Eggshell powder was completely decalcified with EDTA solution, and then DNA was isolated by conventional phenol-chloroform extraction and ethanol precipitation. The efficiency of DNA recovery from eggshells was 50-fold higher than that of a previously reported method. The recovered DNA could be used for PCR, and 10 markers for identifying the Hinai-jidori chicken were detected. The genotypes of the Hinai-jidori exactly matched those of the Hinai-dori breed. Using this method, Hinai-jidori and Hinai-dori eggs could be distinguished from the eggs of Rhode Island Reds. This is the first report of a technique that can be used to discriminate the eggs of Hinai-jidori from those of other chickens, and it can also be utilized to validate the labeling of Hinai-jidori eggs in the market. [source] A nanophosphor-based method for selective DNA recovery in SynthosomesBIOTECHNOLOGY JOURNAL, Issue 7-8 2006Madhavan Nallani Abstract A nanocompartment system composed of an ABA triblock copolymer, where A is poly(dimethylsiloxane) and B is poly(2-methyloxazoline), has been developed for selective recovery and detection of DNA. Translocation of TAMRA-labeled complementary primers into the nanocompartment system has been achieved through two deletion mutants (FhuA ,1,129; FhuA ,1,160) of the channel protein FhuA. Translocation was monitored by fluorescence resonance energy transfer through hybridization of the TAMRA-labeled primer to the complementary sequence of a nanophosphor-DNA-conjugate, which reduces its half-life (FhuA ,1,129, 16.0% reduced; FhuA ,1,160, 39.0% reduced). [source] | ||||||||||