Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.     

Homogeneity studies on human growth hormone

1. Human growth hormone preparations (Raben) have been found to contain 10-40% of denatured growth hormone as shown by radioimmunoassay, reaction of radioiodinated subfractions with antiserum to whole growth hormone, and amino acid analysis, and confirmed by bioassay using the tibia test. 2. The altered fraction was more electronegative than the intact hormone on starch-grain electrophoresis in barbitone buffer, pH8.6. Some heterogeneity of the active material was detectable in simple buffer extracts of a single acetone-stored pituitary gland. 3. The inert fraction was more completely separated from the active hormone as an unretarded fraction from Sephadex G-200 columns with a borate buffer. This separation was due to aggregation of the denatured growth hormone in borate buffer. The active fraction from the Sephadex column still contained some inert material and the amount of this remaining varied considerably between different batches of growth hormone. 4. The radioimmunoassay procedure detects only the immunologically and biologically intact fraction.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Androphilic proteins in cytosols of human benign prostatic hypertrophy.

To examine the properties of androphilic proteins in human benign prostatic hypertrophy, the binding capacity and affinity of the proteins were determined after acetone-treatment, ammonium sulfate precipitation and chromatographies of DEAE and Sephadex G-200. Androphilic proteins in the extract of acetone-dried cytosol from the hypertrophic human prostate was precipitated at 30-50% saturation of ammonium sulfate. The binding of this fraction to dihydrotestosterone and testosterone was high affinity, but the binidng to estradiol-17 beta was the one of non-specific. Androphilic proteins in the 30-50% fraction were eluted from DEAE-cellulose column by buffer containing 0.05 M KCL. On Sephadex G-200 chromatography of 30-50% fraction, the androphilic proteins were observed in three peaks; one was eluted in the void volume and other two were eluted at the sites of IgG and albumin. The amount and ratio of proteins eluted in the void volume and the site of IgG from Sephadex G-200 column were variable in individual tissue samples. The chromatographic behavior of the 30-50% fraction in Sephadex G-200 was not changed significantly by introducing 0.4 M KCl in the system. Polyacrylamide gel electrophoresis was applied for further separation of the proteins.     

Polypeptide antibiotic 26a from Bacillus subtilis. II. Isolation and purification procedures.

Both the analytical and preparative methods by which the preparations of 26a bydrochloride salt with a high antibacterial activity and 20--30% recovery have been obtained from the fermentation fluids of Bacillus subtilis are presented. On an industrial scale the antibiotic can be yielded by absorption of bioactivity on Amberlite CG-50I column and precipitation with picric acid of crude substance from active elutes as adduct which was divided on equilibrated CM--cellulose and finally purified by gel filtration on Sephadex G-25 column. The purified preparation gave a single band by SDS-polyacrylamide gel electrophoresis and one ninhydrin-positive spot by thin-layer chromatography on silica gel G plates corresponding to single zones of bioactivity on bioautograms, and moved as a single peak of almost constant antibacterial activity on Sephadex G-75, G-100 and G-200 columns. The potency of the purest preparations, lot Sephadex G-25, was 6,500--7,000 arbitrary units/mg, and were characterized as follows: purification factor, 57; purity of 98--100% by densitometer scans of SDS-polyacrylamide gels; MIC for Sarcina lutea by twofold agar dilution assay, 0.306 microgram/ml.     

The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]

The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.     

Purification of an anticoagulant from the body fluid of Ascaris suum

An anticoagulant has been purified from the body fluid of Ascaris suum by sequential passage through Sephadex G-50, CM-cellulose and Sephadex G-25 columns then treated with 2 M NaCl, passaged through a Sephadex G-25 column, separated from the phosphate buffer by precipitation of the latter with the CaCl2, then passaged through a Sephadex G-10 column in water. In the body fluid of the worm, the anticoagulant is ionically-bound to a carrier substance. The complex can be split by treatment with 2 M NaCl. The molecular weight of the anticoagulant is slightly less than 1400.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.     

Simple method for the preparation of haptoglobin]

The present paper deals with a method permitting the isolation of haptoglobin 2-2 from human serum or plasma. The haptoglobin is adsorbed to DEAE-cellulose at pH 5.1 by batching. The loaded cellulose is given into a column and the haptoglobin eluted by a 0.1 M to 0.15 M acetate buffer. The last step is a gel-chromatography on Sephadex G-200. Disc- and immunoelectrophoresis were used to test the purity. As by-product acid alpha1-glycoprotein can be obtained.     

Binding specificity of a HeLa DNA-binding protein to DNA and homopolymers

A protein which has affinity for single-stranded DNA but not for double-stranded DNA has been isolated from HeLa cells by DNA-cellulose chromatography. This protein having a molecular weight of 34,000 was accounted for approximately 3% of total soluble proteins. Its binding specificity to DNA and nucleotide homopolymers has been investigated by Sephadex G-200 column chromatography. Specific binding to single-stranded DNA has been confirmed also by this method and furthermore strong binding to poly U has been found.     

Purification of RNA-directed DNA polymerase from mouse spleen infected with Rauscher leukemia virus.

RNA-directed DNA polymerase was purified from spleens of Balb/c and NMRI mice infected with Rauscher murine leukemia virus. The method includes cell fractionation and lysis of microsomal fraction, chromtography on Sephadex G-200 and phosphocellulose. Estimation of molecular weight from the sedimentation rate of the purified enzyme in a glycerol gradient was consistent with a structure containing one polypeptide with a molecular weight of 70,000. Purified RLV DNA polymerase from spleen could transcribe purified DNA polymerase from purified virions. This simple preparation method offers a procedure for large scale preparation of the RNA-directed DNA polymerase which can be used for synthesis of DNA complementary to mRNA.     

Technical note: improved ancient DNA purification for PCR using ion-exchange columns

A novel method of ancient DNA (aDNA) purification was developed using ion-exchange columns to improve PCR-amplifiable DNA extraction from ancient bone samples. Thirteen PCR-resistant ancient bone samples aged 500-3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification. We suggest that the ion-exchange column-based method will be useful for the improvement of PCR-amplifiable aDNA extraction, particularly from the poorly preserved, PCR-resistant, ancient samples.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Simple radioassay for uridine phosphorylase and 5'-nucleotidase

A method is described whereby a large number of chromatographic fractions containing protein or enzyme components may be run on a single-starch gel. A multiple punch for making the sample holes, gel slab support, and a gel slicer used to cut several slices from the one gel for staining for the various enzyme or protien constituents are described. An example is given of a run using fractions obtained from a Sephadex G-200 column chromatography run of a herring muscle extract. Densitometry may be used to assay the stained gels. In another example a commercially obtained muscle lactate dehydrogenase preparation was chromatographed on a Sephadex G-200 column. The electrophoretic run of the fractions revealed the presence of both heart-type A and muscle-type B lactate dehydrogenase subunits and showed that the column run had effected a partial separation of the various tetramers.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

Heterogeneity of big-big hPRL in hyperprolactinemia

Sera from a patient with macroprolactinoma (case 1) and from a hyperprolactinemic woman with regular menstruation (case 2) were analyzed for prolactin activity by gel filtration using Sephadex G-100, Sephadex G-200 and TSK G3000SW columns. The chromatographic profile by Sephadex G-100 showed that the percentage of immunoreactive big-big hPRL was 10.7% in case 1 and 64.1% in case 2. On Sephadex G-200 and TSK G3000SW columns, the molecular weight of big-big hPRL was estimated to be more than 500,000 daltons (big-big1 hPRL) in case 1 and approximately 250,000-300,000 daltons (big-big2 hPRL) in case 2. Big-big1 hPRL in case 1 was converted to big and little hPRLs when the serum was treated with 2-mercaptoethanol (2-ME), but part of the big-big2 hPRL in case 2 was converted to a larger molecule. Radioactive big-big hPRL generated by mixing labeled hPRL with the serum from case 1 was eluted with the void volume on Sephadex G-100 column and was not converted to the other molecular forms after 2-ME treatment. There were two radioactive big-big hPRL on TSK G3000SW column and these estimated molecular weights were more than 300,000 daltons. The data demonstrated the existence of at least two forms of big-big hPRL in the serum and indicated that radioactive big-big hPRL may be different from these hPRLs in the serum.