Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material

Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.     

Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide

Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.     

Use of silica gel polymer for DNA extraction with organic solvents

Phenol and chloroform are the standard solvents used for DNA extraction. These solvents aid in the removal of protein and lipid from crude or partially purified cell extracts. Although the procedure is well established, the solvents are noxious, caustic, and unpleasant. We describe in this paper the use of a special blood collection tube to isolate the offensive organic solvents. With the use of silica gel polymer containing tubes, phenol, phenol:chloroform, or chloroform can be separated from the DNA containing aqueous phase in a rapid and safe manner. The method permits higher yields of DNA since the DNA is poured from the tube rather than aspirated with pipet.     

Recovery of mitochondrial DNA from blood leukocytes using detergent lysis

Mitochondrial DNA (mtDNA) was isolated from leukocytes contained in whole blood of cattle. Leukocyte membranes except the nuclear envelope were solubilized in a buffer that contained 1% Triton X-100. After sedimentation of cell nuclei, mtDNA was purified from the cell lysate by organic solvent extraction and ethanol precipitation. Approximately 5 µg of mtDNA was recovered from 400 ml of whole blood, a quantity sufficient for routine DNA cloning procedures or for detailed restriction mapping studies. mtDNA isolated with this method is a suitable substrate for several DNA-modifying enzymes. Thus, preparation of mtDNA from blood by detergent lysis provides a noninvasive alternative to tissue biopsy for characterization of mitochondrial genotypes in studies of evolutionary genetics and population dynamics.This work was supported in part by the Iowa State University Biotechnology Council and by the Holstein Association.Journal Paper No. J-13683 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2736.     

Extraction of mitochondrial membrane proteins into organic solvents in a functional state

Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1-79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein-lipid complexes, are fairly stable in organic solvents; in a month of storage at 4 degrees C in hexane some enzymes loose less than 50% of their activity.     

Supercritical carbon dioxide extraction of griseofulvin from the solid matrix obtained after solid-state fermentation

Globally there is an increasing concern to minimize the use of organic solvents, particularly the chlorinated ones because of their suspected human carcinogenicity. The use of ecofriendly carbon dioxide as an alternative to organic solvents would be appropriate in the perspective of green technology. Supercritical carbon dioxide (SC-CO(2)) extraction is suitable for extraction of nonpolar compound with molecular weights less than 400. Griseofulvin is an antifungal antibiotic having a molecular weight of 353, making it amenable to SC-CO(2) extraction. This work brings out the potential of supercritical carbon dioxide extraction (SCFE) for downstream processing of griseofulvin from the solid matrix obtained after solid-state fermentation (SSF). The optimized conditions for SCFE of griseofulvin from dried media after SSF were a flow rate of 0.4 L/min, temperature of 60 degrees C, and contact time of 90 min (30 min static + 60 min dynamic) at a pressure of 450-455 bar.     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

Solid phase microextraction gas chromatographic analysis of organophosphorus pesticides in biological samples

Headspace-solid phase microextraction (HS-SPME) was studied and optimised for the determination of four common organophosphorus pesticides (OPPs) in biological samples. Various parameters controlling SPME were studied: choice of SPME fiber, type and content of salt added, preheating and extraction time, desorption time, extraction temperature. Capillary gas chromatographic analysis with nitrogen phosphorus detection (GC-NPD) facilitates sensitive and selective detection of the OPPs: malathion, parathion, methyl parathion and diazinon. Fenitrothion was used as the internal standard. The method was applied to the determination of the pesticides in human biological specimens: whole blood, blood plasma, urine, cerebrospinal fluid, liver and kidney. Limits of detection ranged from 2 to 55 ng/ml depending on pesticide and type of specimen. The developed methodology overcomes limitations and obstacles of conventional methods such as the use of organic solvents, the formation of emulsions and the tedious-cumbersome procedures. The proposed protocol is seen as an attractive alternative to be used in routine toxicological analysis.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

Batch solvent extraction of flavanolignans from milk thistle (Silybum marianum L. Gaertner)

Seeds of milk thistle (Silybum marianum L. Gaertner) contain silymarins and ca. 25% (w/w) of oil. A pre-treatment step involving refluxing with petroleum ether is usually performed before extraction of the silymarins using organic solvents. This paper compares the extraction of whole and defatted milk thistle seeds in various solvents as a function of temperature. The extraction of whole seeds of milk thistle with water at 50, 70 and 85 degrees C was also examined: the yield of silymarin increased with increasing water temperature. In most cases, ethanol at 60 degrees C recovered the largest quantities of silymarins. However, boiling water proved to be an efficient extraction solvent for the more polar silymarins such as taxifolin and silychristin, even when using whole seeds. Extractions of defatted seed meal with boiling ethanol returned maximum yields of 0.62, 3.89, 4.04, and 6.86 mg/g defatted seed of taxifolin, silychristin, silybinin A and silybinin B, respectively. When extracting defatted seed meal with ethanol, yields of taxifolin, silybinin A and silybinin B were, respectively, 6.8-, 0.95-, 1.7- and 1.6-fold higher than when extracting whole seeds. When extracting with boiling water, the yields of silychristin, silybinin A, and silybinin B were 380, 47 and 50% higher for whole seeds compared with defatted seeds.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method

In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS) and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.     

Immobilization of enzymes with polyaziridines.

A novel method of enzyme immobilization using a low molecular weight prepolymer of tri-functional aziridines which can immobilize enzymes both by covalent attachment and entrapment within a gel matrix is described. The enzymes are immobilized on a solid support and exhibit an excellent retention of enzymatic activity. The immobilization procedure is essentially a single step process which can be easily performed at room temperature or 4 degrees C in either aqueous solution or in an inert organic solvent. The polyaziridines used in the immobilization are nontoxic, available in bulk at low cost and completely miscible with water and many organic solvents, thus providing one of the most satisfactory methods of immobilization available.     

Enhancing the biocatalytic potential of carbonyl reductase of Candida viswanathii using aqueous-organic solvent system

In the present work, the toxic effect of various solvents with different Log P values was studied on the whole cells of Candida viswanathii. Experiments showed that the lower concentrations of some solvent increased both the activity retention and enzyme activity as compared to the control while this was not the case with higher concentrations of the same solvents. The model compound taken in the present study was 1-acetophenone. The percentage conversion improved from 76 to 94%. Addition of 2-propanol increased the substrate tolerance, giving the conversion of 90% compared to 9% in control at a substrate concentration of 70 mM in 1h. The operational stability increased at higher temperatures with the addition of 2-propanol in the reaction mixture with good conversion (90%) and enantiomeric excess (>99%) at 45 degrees C and 50 degrees C. The effect was also found to be prominent in other tested substrates. In order to further stabilize the cells for long term use in higher concentration of organic solvents, the cells were further immobilized, and were found to have higher activity retention than that of free cells.     

Biocatalysis using an organic-soluble enzyme for the preparation of poly(lactic acid) in organic solvents

Proleather from Bacillus sp. was chemically modified with decanoyl chloride for enhanced activity for the preparation of poly(lactic acid) in organic solvents. The modified enzyme was highly soluble (up to 44 mg-protein/ml) and active in various organic solvents including chloroform, tetrahydrofuran (THF), pyridine and acetone. The organic-soluble proleather efficiently catalyzed the polymerization of ethyl lactate. The reaction rate was 4-22 times that of native proleather, depending upon the solvent applied. The solubilized enzyme showed a highest activity at 50 degrees C, the same optimum temperature for both the native proleather and an immobilized enzyme, Novozyme-435. Denaturation of the enzymes' protein structures appeared to be the critical factor regulating the optimum activity temperature. Differential scanning calorimetry (DSC) analyses of the enzymes showed endothermic peaks around 55 degrees C, indicating the proteins' structures altered in that temperature range. Interestingly, the activity of the solubilized enzyme showed a more complicated water dependence as compared to native proleather.     

Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.