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PubMed1993

Chromosomal DNA extraction after restriction endonuclease treatments: a study by microdensitometry and electrophoresis.

The appearance of G- and C-banding patterns on metaphase chromosomes from the Don cell line (Cricetulus griseus) was accompanied by extraction of chromosomal DNA. A microdensitometric technique was employed to measure the loss of DNA produced by Hin dIII, Hae III and Eco RI endonuclease digestion on fixed chromosomes, from 0.5 to 24 h of treatment. Agarose gel electrophoresis was carried out to estimate the size of the DNA fragments extracted after digestion by Alu I and the restriction endonucleases already mentioned. Results obtained show that Alu I and Hae III, which possess 4 base pair recognition sequences, caused higher DNA extraction than enzymes with recognition targets of 6 base pairs (Hin dIII and Eco RI). Incubation buffers induced G-banding patterns which were accompanied by DNA extraction, although this loss was lower than that produced by endonuclease treatments.

Animals↗
PubMed1992

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.

DNA↗
PubMed1997

Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction.

Malaria remains a disease of underdeveloped and remote regions of the world. The application of polymerase chain reaction (PCR) technology to malaria epidemiology has the potential for increasing our knowledge and understanding of this disease. In order to study malaria in all geographical locations it is important that specimen collection and DNA extraction for PCR be kept simple. Here we report a method for extracting DNA from dried blood spots on filter paper which is capable of detecting one Plasmodium falciparum and two Plasmodium vivax parasites/microliter of whole blood by nested PCR without compromising the simplicity of specimen collection or DNA extraction.

Animals↗
PubMed1975

Relationships between non-extractable DNA and the bacterial growth cycle.

Escherichia coli DNA has been fractionated into extractable and non-extractable DNA after deproteinization of detergent-lysed cell preparations with chloroform-isoamyl alcohol. The former was extracted with dilut buffered saline whereas the latter remained in the interphase layer associated with residual cellular debris from which almost 40 percent could be released by incubating with pronase. About 20-25 percent more amino acid residues were bound to the pronase-released DNA than to the extractable DNA, but the relative distribution of the residues in the two DNA samples was virtually identical. The specific activities and the relative amounts of denser (1.709g-cm-3) and lighter (on the surface of CsCl gradients) DNA fractions from E. coli, grown in the presence of labeled thymidine, indicated that these two corresponded to extractable and non-extractable DNA, respectively. The relative amounts of the two fractions varied with the growth phase primarily as a function of the growth rate. Age and metabolic state of cells in the culture or those used as inocula could modify this relative distribution. When growth rate was maximal, the ratio of the two remained at about 1. During lag phase when no appreciable net synthesis of DNA could be detected, there was a rapid and preferential incorporation of labeled thymidine into non-extractable DNA. A disproportionate increase in the fraction of the total DNA which was extractable, was also abserved but only when stationary phase cultures were used as inocula. Complete equilibration of the label in the two DNA fractions was attained only after cultures had reached mid-log phase of growth. Similar results were obtained when prelabeled cells were used. These data have been interpreted as suggesting that the rate of cell growth and DNA synthesis are related to the number or size of sites of attachment of DNA to some cellular structure. Newly synthesized DNA would be attached to different sites on this structure and initiation of DNA replication in lag phase would require reorientation of the two kinds of DNA. Small peptides which are firmly bound to the DNA and which vary quantitatively with the rate of DNA synthesis could perhaps be involved in the attachment to the sites.

Amino Acids↗
PubMed1994

ScreenMax plasmid mini-prep: super rapid plasmid DNA extraction method.

An ultra-quick method for plasmid DNA extraction using the ScreenMax Plasmid Mini-prep Kit was optimized. Since the method has fewer steps than current methods, the entire process takes only 12 min for two sets of plasmid DNA extractions. DNA quality was excellent for further analyses, including DNA sequencing. The kit's new medium, MMB, provided 5 times the viable cell count and 7 times the dry cell weight compared with the conventional LB medium. The yield of plasmid DNA was 5 times better than from LB medium. From 200 microL of culture, the maximum yield was 7.5 micrograms. The ScreenMax Plasmid Mini-Prep is not only simple and economical but also safe because of its mild reagents. This new procedure is both reliable and reproducible.

DNA↗
PubMed2005

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR.

To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.

Cell Wall↗
PubMed2005

An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue.

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% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. CONCLUSION: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies.

DNA, Neoplasm↗
PubMed1996

PCR DNA typing of stamps: evaluation of the DNA extraction.

Today, the PCR analysis of DNA from the saliva deposited on a stamp of an anonymous letter can lead to an identification. However, this analysis still involves problems, and DNA extraction can be particularly difficult. A comparative study of two DNA extraction methods with two categories of postage stamps was carried out and a purification process was tested. This study shows that the extraction with phenol/chloroform gives much better results than Chelex extraction. A purification process such as the use of Centricon 100 microconcentrators is recommended when an inhibition of PCR is present. This operation, however, results in a large loss of material.

DNA↗
PubMed2005

Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments.

AIMS: To compare three bacterial DNA extraction procedures on cold-smoked salmon (CSS) and assess the impact on their efficiency of two physical treatments of the food matrix, ionizing irradiation and freezing. METHODS AND RESULTS: As molecular methods for bacterial detection have become an important analytical tool, we compared bacterial DNA extraction procedures on CSS. Working with frozen and irradiated CSS, we obtained negative responses from samples known to be highly contaminated. Thus, we decided to study the impact of these two physical treatments on bacterial DNA extraction procedures. The efficiency of bacterial DNA extraction directly from the fish matrix suspension was measured by an rpoB PCR-based reaction. The results demonstrated that the DNeasy tissue extraction kit (Qiagen, Courtaboeuf, France) was the most efficient and reproducible method. We also showed that freezing and ionizing irradiation have a negative impact on DNA extraction. This was found probably not to be due to inhibition as the PCR reaction remained negative after adding BSA to the PCR mix reaction. CONCLUSIONS: The extraction kit was the most efficient method. Physical treatments were shown to hamper bacterial DNA extraction. SIGNIFICANCE AND IMPACT OF THE STUDY: Attention must be paid to molecular bacterial detection on food products subject to freezing or to ionizing irradiation.

Animals↗
PubMed1997

Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures.

The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.

AIDS-Related Opportunistic Infections↗
PubMed1975

Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles.

DNA extracted from Dane particles has been characterized by gel electrophoresis and restriction enzyme cleavage with endonuclease R-HaeIII (from Hemophilus aegyptius). Dane particle DNA is proposed to be a double-stranded circular DNA approximately 3600 nucleotides in length containing a single-stranded gap of 600-2100 nucleotides. The endogenous DNA polymerase (DNA nucleotidyl-transferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) reaction appears to repair this single-stranded gap.

DNA Nucleotidyltransferases↗
PubMed1998

DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods.

The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa-stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze-thaw method, boiling in 10% Chelex-100 resin solution, proteinase K/Tween 20/NP-40 method coupled with simplified phenol/ chloroform/isoamyl alcohol protocol or salting-out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen. Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa-stained bone marrow slides.

Azure Stains↗
PubMed1990

A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

Blood Cells↗
PubMed2003

DNA extraction from bronchial aspirates for molecular cytology: which method to take?

OBJECTIVE: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods. METHODS: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated. RESULTS: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of beta-globin gene fragments (268, 536, and 989 bp) was 100%. CONCLUSION: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR-based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.

Adult↗
PubMed2003

A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit.

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.

DNA↗
PubMed1995

A simple method for extracting DNA from old skeletal material.

Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.

Acetates↗
PubMed2001

An improved method for DNA extraction from paraffin sections.

The acquisition of comparable quality and quantity of DNA extracts is the prerequisite to the success of comparative genetic analyses. Although several DNA extracting protocols on paraffin sections have been introduced, the importance of deparaffinization, the procedure for obtaining an adequate hematoxylin staining, the significance of the ratio of the cell number to the enzyme volume, and a practical means for monitoring the digestion process have not been sufficiently addressed. These, however, are the most important factors accountable for a failure of DNA extraction. To minimize the impact of these factors, we have developed several unique strategies, including: (1) incubating sections at 80 degrees C for 30-60 minutes prior to xylene treatment, (2) checking each section to insure the complete removal of paraffin; (3) treating hematoxylin stained sections or cells with de-staining solutions; (4) using a micrometer inserted into the eyepiece of a microscope to estimate the number of cells collected and adjusting the enzyme volume according to the cell number; and (5) monitoring the digestion process with a magnifier. With these strategies, we have been able to consistently obtain comparable quality and quantity of DNA extracts which yielded uniform PCR products regardless of variations in tissue embedding and processing.

Clone Cells↗