Recovery of amino acids and tRNA after scintillation counting of radioactive aminoacyl-tRNA in filter paper strips

Amino acids are quantitatively recovered from aminoacyl-tRNA in filter paper strips by treatment with 0.3 m ammonium carbonate. A simple apparatus is described for deaminoacylation and separation of amino acids and tRNA. The amino acids, thus recovered, can be directly analyzed by paper chromatography since ammonium carbonate is removed by lyophilization. Nonvolatile reagents such as Tris-HCl and sodium carbonate also hydrolyze aminoacyl-tRNA, but unless the products of deaminoacylation are desalted, the presence of these reagents gives rise to chromatographic artifacts such as peak broadening, appearance of multiple peaks, displacement of R_f values, etc.tRNA remaining after deaminoacylation retains only a small fraction of its original capacity to bind amino acids. Part of this loss of activity appears to be due to structural damage to tRNA in the course of washing and counting procedures and in part, due to the effect of filter paper in shifting the equilibrium of the reaction toward hydrolysis of aminoacyl ester bond.     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.     

High-Yield and Phylogenetically Robust Methods of DNA Recovery for Analysis of Microbial Biofilms Adherent to Plant Biomass in the Herbivore Gut

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the ??repeated bead beating and column (RBB+C) purification?? method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions.     

Rapid and inexpensive recovery method of DNA fragments from agarose and polyacrylamide gels by a cotton-wool column tube.

We developed a rapid, convenient, simple, and inexpensive method for isolating pure DNA from agarose and polyacrylamide gels using cotton wool tubes. DNA fragments ranging in size from 193-23,130 bp can be easily recovered within 2 hours by centrifugation through cotton wool from gel slices. The recovery rate of this method is 35% to 50%, when estimated for isolation of lambda DNA-HindIII fragments. We have also recovered 700-bp polymerase chain reaction (PCR) products using cotton wool tubes from electrophoresis on both a 0.8% agarose gel and a 6% polyacrylamide gel, in which satisfactory yields of more than 50% were obtained. The DNA thus recovered in this way is biologically active and can be used as a substrate for further experimental procedures without additional purification steps.     

Detection of ionizing radiation-induced DNA double-strand breaks by filter elution is affected by nuclear chromatin structure

Chinese hamster ovary cells were irradiated with 250 kVp X rays and analyzed for the presence of DNA double-strand breaks using either polycarbonate filter elution or pulsed-field agarose gel electrophoresis at neutral pH. Reduction in DNA length detected by filter elution was produced as a nonlinear function of increasing radiation dose, with a quasi-threshold at low total dose, and as a first-order function of increasing radiation dose as detected by gel electrophoresis. The quasi-threshold observed with filter elution was eliminated when nuclei were isolated from irradiated cells and their chromatin relaxed in a buffer containing low-molarity monovalent cation prior to analysis by filter elution. The results suggest either that the chemical structure of the DNA double-strand breaks produced by low-LET radiation necessitates a DNA relaxation step before they can be detected accurately by filter elution, or that at low total radiation dose a DNA complex forms on the polycarbonate filter.     

Sequence Analysis of Marine Virus Communities Reveals that Groups of Related Algal Viruses Are Widely Distributed in Nature

Algal-virus-specific PCR primers were used to amplify DNA polymerase (pol) gene fragments from geographically isolated natural virus communities. Natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic peninsula. Genetic fingerprints of algal virus communities were generated using denaturing gradient gel electrophoresis (DGGE). Sequencing efforts recovered 33 sequences from the gradient gel. Of the 33 sequences examined, 25 encoded a conserved amino acid motif indicating that the sequences were pol gene fragments. Furthermore, the 25 pol sequences were related to pol gene fragments from known algal viruses. In addition, similar virus sequences (>98% sequence identity) were recovered from British Columbia and Antarctica. Results from this study demonstrate that DGGE with degenerate primers can be used to qualitatively fingerprint and assess genetic diversity in specific subsets of natural virus communities and that closely related viruses occur in distant geographic locations. DGGE is a powerful tool for genetically fingerprinting natural virus communities and may be used to examine how specific components of virus communities respond to experimental manipulations.     

The Usefulness of DNA Sequencing After Extraction by Whatman FTA Filter Matrix Technology and Phenotypic Tests for Differentiation of Candida albicans and Candida dubliniensis

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.     

The cleavage of Drosophila melanogaster DNA by restriction endonucleases

Drosophila melanogaster DNA, together with λ and E. coli DNAs as controls, was digested with three different restriction endonucleases: EcoR_I, Hind, and Hae. The size distributions of the segments were characterized by gel electrophoresis. More than 85% of the D. melanogaster DNA was found in a broad distribution of segment lengths consistent with random location of restriction sites. However, some DNA was spared and recovered in very long (≥20500bp) segments. These segments proved to be mostly simple sequence DNA. No complex spared segments could be found in Hind and Hae digests, while 50% of the spared EcoR_I segments had a complexity exceeding that of the E. coli DNA spared by this enzyme. These data do not support the hypothesis that chromomeres contain long regions of purely tandemly repeating sequences.     

Filter elution assays for DNA damage: practical and mechanistic significance of the DNA in the filter support wash.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.     

Preservation of encysted polytomella.

Cysts of Polytomella parva and Polytomella caeca were recovered after 7 days storage at cryogenic temperatures following drying on shredded filter paper, silica gel or without added substrate. Accelerated storage testing, by exposing dried material to elevated temperatures, indicated that shredded filter paper was the best of the substrates tested. Polytomella parva was recovered after 5 years storage at -30 degrees C when dried on filter paper but not when dried on silic agel. Determinations of the number of cysts recovered indicated that viable cysts survived all conditions of storage tested. However, excystment following storage was delayed, the extent depending on storage conditions and the substrate used for drying. Most rapid recovery occurred when cysts were rehydrated immediately after drying, and after storage on filter paper at below -70 degrees C.     

Microelectrophoretic method for the detection of proteinase inhibitors

A microelectrophoretic method for the detection of proteinase inhibitors is deseribed. Microscope slides covered with agar gel containing casein were used for the electrophoretic separation of proteins from bean seeds. Subsequently, the slides were covered with a filter paper strip saturated with a proteinase solution of a known concentration. After 60 min of digestion, the filter paper strips were removed, and the slides were stained with amido black. Blue spots appeared where the casein substrate was protected from digestion by the presence of proteinase inhibitors. Single seed or leaf extracts can be studied by this method. Different trypsin inhibition patterns were observed for samples of different bean varieties. An inhibitor for subtilisin was detected in all the Ph. vulgaris samples studied.     

Electroelution of DNA and protein from polyacrylamide and agarose gels

An electroelution method is described for the recovery of DNA and protein from agarose or polyacrylamide gels. The samples to be electroeluted are compartmentalized in a modified microcentrifuge tube fitted with dialysis membranes. This procedure is simple, rapid, inexpensive and efficient. Within 30 min to 2 hrs, the recovery of the sample is nearly quantitative. DNA fragments recovered can be directly subjected to DNA sequence analysis or enzymatic reactions after ethanol precipitation. Proteins can also be recovered after separation by acrylamide gel in the presence or absence of detergents and be ready for further analysis.     

Newborn screening by DNA analysis of dried blood spots

Summary Amplification of DNA recovered from a dried blood spot was used to genotype individuals with sickle cell disease, sickle cell carriers, and controls. A single 200-l blood spot applied to a filter paper provides sufficient material for more than 20 genetic analyses. In addition, the stability of the DNA is such that adequate material for amplification can be isolated from dried blood spots up to a year following collection. The DNA analysis methods described in this study could be applied to large-scale screening of newborns for genetic disorders.     

A rapid preparation of plasmid DNA fromSaccharomyces cerevisiae

A procedure for extraction of plasmid DNA from Saccharomyces cerevisiae is described. The plasmid DNA of interest is extracted together with 2-micron circular DNA naturally occurring in many yeast strains. Spheroplasts are lyzed at alkaline pH which denatures linear but not covalently closed circular (CCC) DNA. The CCC DNA is recovered by ethanol precipitation and can be detected by gel electrophoresis or used for routine bacterial transformation.     

A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels.

Defined RNA fractions can be recovered from low gelling temperature agarose gels by a combination of agarose melting at 65°C and phenol extraction. By this approach RNA molecules up to a size of 37 kb can be eluted undegraded with a recovery of 60–90%. The method is applicable also to DNA and the eluted DNA can be correctly cleaved with restriction endonucleases as shown for λDNA using EcoRI.     

Rapid mapping of restriction sites of a DNA: restriction of agarose gel and two-dimensional analysis of end-labeled DNA.

A new two-dimensional technique for the mapping of restriction sites is presented. Linear DNA labeled at both ends is first partially digested with the restriction endonuclease for which a map is desired. Following electrophoresis of the partial digest in an agarose gel, complete digestion of the fragments in the gel matrix with a second restriction enzyme is carried out. Electrophoresis in the second dimension resolves two sets of labeled spots: one set from the left and the other from the right end. For a given band of the autoradiogram of the first dimension gel, the mobility of the band gives the size of the DNA fragment, and therefore the distance of a particular restriction site from one of the ends of the original linear DNA. The mobility of the labeled spot derived from this band in the second dimension gel allows one to distinguish whether the distance deduced above is from one end or the other. Additional information about the location of one set of restriction sites for one enzyme relative to those for a second enzyme can also be obtained using the two-dimensional method. The advantages of the technique are the small amount of DNA required and the rapidity with which many maps can be constructed from one labeled DNA. As a test of the method, maps for the HindIII and HaeIII cleavage sites of circular phage PM2 DNA have been obtained, after first converting the DNA to the linear form by digestion with HpaII.     

Construction of a circular single-stranded DNA template containing a defined lesion

We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication assays.     

A rapid two-dimensional fractionation of DNA restriction fragments

Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme. The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments. This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.     

Restriction endonuclease mapping by crossed contact hybridization: the ribosomal RNA genes of Achlya ambisexualis.

A rapid, convenient and economical method for the hybridization of electrophoretically resolved RNA to DNA restriction fragments immobilized on nitrocellulose filters is described. DNA was digested, electrophoresed on agarose gels in a wide band and transferred to a nitrocellulose filter. The filter was then placed on the surface of a second gel containing radioactively labeled RNA electrophoresed under denaturing conditions in a similar way. The filter and gel were oriented so that the DNA and RNA bands were perpendicular to one another and the RNA was transferred from the gel through the filter under conditions which promote RNA-DNA hybridization. Following washing, the filter was autoradiographed. RNA-DNA sequence relationships could be conveniently determined from the spots produced at regions of intersection of homologous nucleic acids. The two dimensional array formed in this procedure fascilitates the rapid ordering of DNA restriction fragments. An example of its use for this purpose is presented.