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"Chelex 100" chelating resin has been previously proposed for the rapid extraction of human DNA for polymerase chain reaction. Protocols are given for the rapid extraction of bacterial and viral DNA from cultures or clinical samples. The DNA samples obtained were suitable for use in polymerase chain reaction.
Apolipoprotein E (APOE) genotyping of genomic DNA extracted from formaldehyde-fixed specimens is cumbersome: there is not only a low yield or failure of PCR amplification (presumably due to degradation of DNA in the formaldehyde-fixed and paraffin-embedded tissue), but the standard method also involves the separation of DNA fragments as small as 48, 72, 81 and 91 bp requiring high-yield PCR products. Here we report about a semi-nested PCR method suitable for providing specific high-yield PCR products from DNA that has been extracted from formaldehyde-fixed specimens which initially generate low-quality templates. This method facilitates reliable APOE genotyping of DNA from difficult templates.
A modification to the DNA extraction method "preferential lysis" (Gill et al. 1985) is proposed which can be applied to DNA mixtures of vaginal cells and spermatozoa. In mixtures with a low sperm content the further loss of sperm DNA caused by the extraction can be avoided by using "mild preferential lysis". Amplification by PCR (polymerase chain reaction) then yields sufficient DNA to be able to identify both components in the mixture.
Accurate enumeration of indicator organisms such as Escherichia coli is important for assessing the safety of water and wastewater samples. Recent research has shown that E. coli can enter a viable but non-culturable state; therefore, traditional cultivation methods could potentially underestimate the quantities of the organisms. The goals of the research were to develop and verify a DNA extraction protocol and a quantitative polymerase chained reaction (PCR) method for E. coli enumeration in digested biosolids. A solvent-based DNA extraction protocol with extensive cell lysis recovered approximately 78-84% of spiked DNA. In comparison, a commercial kit only recovered 28-42% of DNA, likely from inefficient cell lysis. The developed competitive touchdown PCR (cPCR) method for E. coli enumeration was comparable to both real-time PCR (rt-PCR) and cultivation methods with sensitivity of approximately 50,000-500,000 E. coli per gram dry solids (DS), which is suitable for Class B biosolids monitoring in the US and "conventional" biosolids in the European Union. The cPCR protocol provides a less expensive alternative than the rt-PCR as a culturing independent method for enumerating E. coli.
The focus of the present investigation was to study, via molecular biology techniques, the character of the DNA present in individual archival celloidin-embedded human temporal bone sections. Polymerase chain reaction (PCR) amplification of 92 base pair (bp), 121 bp, and 471 bp regions of mitochondrial DNA (mtDNA) extracted from a single archival celloidin-embedded human temporal bone section was used to assess the length of the template DNA extracted. The effects of digestion time and sample motion during the extraction method on DNA concentration was also studied. These data are crucial to determine the limits of applying PCR technology to amplify specific genomic DNA targets located within the human inner ear. Further development of these methods will allow additional molecular temporal bone pathologic studies to be completed and, more specifically, hypotheses regarding the molecular etiopathogenesis of many auditory, vestibular, and facial nerve disorders, such as autoimmune hearing loss, congenital hearing losses, Meniere's disease, otosclerosis, or Bell's palsy could be tested. The results described should be of great value to those investigators extracting DNA from archival individual human temporal bone sections for PCR assays of specific genetic alterations or infectious agents associated with temporal bone pathologies.
Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100 degrees C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.
The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.
The automated MagNA Pure DNA extraction method for Chlamydia trachomatis was compared with the manual Cobas Amplicor protocol using 100 microL of input sample volume from 964 specimens. Agreement between protocols was 96.1%. The automated extraction method had a sensitivity of 99% and a specificity of 100%. Amplification inhibition observed after manual preparation of samples (3.8%) was not apparent following automated extraction. Using 200 microL of sample in the automated extraction process lowered the detection limit without raising the inhibition rate. Furthermore, the automated extraction method halved the hands-on time required for the procedure.
Several schemes of acidic extraction of RNA taken from biochemical or cytochemical literature were compared by means of ultraviolet and visible cytospectrophotometry. Basing on the data obtained an extraction scheme has been recommended which consists in the treatment of nervous tissue sections with 16 per cent perchloric acid at 4 degrees C for 48 h. This extraction has been shown to extract completely cellular RNA without extracting DNA and proteins from the nervous tissue cells. Using this scheme, ultraviolet cytospectrophotometry of neurons and glial cells was performed twice, before and after the selective RNA extraction, which enabled a calculation of RNA concentration by the difference of the absorption values. In mice, after 3 h-swimming, and in cats, after 2-week daily injections of adrenaline, a number of quantitative and qualitative RNA changes were found out in nerve and glial cell of mouse spinal cord and cat sympathetic ganglion with the aid of the recommended scheme of RNA extraction.
Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.
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.
In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less than 1% of bacteria visible microscopically can be cultured. Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments (the Ogram and Tsai and methods [A. Ogram, G. S. Sayler, and T. Barkay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B. H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 1991]) and those in which cells are removed from sediments prior to lysis (the Jacobsen method [C. S. Jacobsen and O. S. Rasmussen; Appl. Environ. Microbiol. 58:2458-2462, 1992]). DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods. However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields.(ABSTRACT TRUNCATED AT 250 WORDS)
DNA was serially studied in 20 samples of buffy coat stored at room temperature. Each sample was divided into 5 equal volumes, namely D0, D3, D5, D7 and D10. DNA extraction was performed on days 0, 3, 5, 7 and 10 after blood collection. The mean ratio of OD260/OD280 of the DNA obtained from D0 to D10 ranged from 1.77 to 1.79, and the mean amounts of the DNA obtained from D0 to D10 ranged from 602 to 740 ng/ul. There were no significant differences in the mean ratio and amounts of DNA obtained among these samples (p > 0.05). Subsequently, amplification was successfully performed from this template DNA to yield products of 1.4 kb and 142 bp at the sites associated with beta globin and factor VIII genes, respectively. These findings suggest the possibility of sending blood samples for DNA analysis by mail, or no ice is required during transportation.
Homalodisca coagulata Say (Hemiptera: Cicadellidae) is a major agronomic pest because it transmits Xylella fastidiosa (Wells), the bacterium that causes Pierce's disease of grapevine. The ability to easily detect X. fastidiosa in populations of H. coagulata facilitates epidemiological studies and development of a monitoring program supporting disease management. Such a program depends on a detection protocol that is rapid, reproducible, and amenable to large sample sizes, while remaining sensitive enough to detect low amounts of pathogen DNA. In this study, we developed an improved method to speed DNA extraction by implementing a simple vacuum step that replaces labor- and time-intensive maceration of tissue samples and that is compatible with manufactured DNA extraction kits. Additionally, we have developed a SYBR Green-based real-time (RT)-polymerase chain reaction (PCR) system, which uses traditional PCR primers that are relatively inexpensive and effective. Using this extraction/RT-PCR system, we found no statistically significant differences in the detection of X. fastidiosa among samples that were either immediately extracted or stored dry or in mineral oil for 10 d at -4 degrees C. In further testing, we found no significant reduction in detection capabilities for X. fastidiosa-fed H. coagulata left in the sun on yellow sticky cards for up to 6 d. Therefore, we recommend a field-based detection system that includes recovery of H. coagulata from sticky traps for up to 6 d after trapping, subsequent freezing of samples for as long as 10 d before vacuum extraction is performed, and detection of the bacterium by SYBR Green-based RT-PCR.
Organic and Chelex extraction methods are compared to five commercially available DNA extraction kits. For forensic purposes, comparison of different extraction strategies is based on the ability of the methods to economically recover high yields of amplifiable DNA from small blood samples in a quick, simple and safe manner. The findings of this study indicate that resin-based methods, such as Chelex, are best able to meet these criteria.
Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.
A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.
Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.