Nucleic Acid Digestion Research Articles

Identification of four novel developmentally regulated gamma hemoglobin mRNA isoforms

In the course of developing assays for the molecular prediction of biological age, we serendipitously discovered four novel isoforms of gamma hemoglobin mRNA, designated HBG1n1, HBG1n2, HBG2n2, and HBG2n3, collectively termed HBGn isoforms. Here we report the molecular characterization and tissue expression of these isoforms. RNA obtained from human peripheral blood and various fetal and adult tissues was amplified with duplex reverse transcription polymerase chain reaction (RT-PCR) assays to determine the expression profiles of the HBGn isoforms. To determine if their molecular origin was either a genomic recombination or an undefined RNA rearrangement event, DNA and RNA samples were pretreated with RNaseI and/or DNaseI and then analyzed with the duplex RT-PCR assays. Alignment analysis indicated that the HBG1n(1/2) and HBG2n(2/3) isoforms were identical to the expected parental HBG1 or HBG2 sequences except for unique deletions that spanned the 3' end of exon two, the entire second intron, and the 5' end of exon three. RT-PCR duplex reactions revealed that the isoforms exhibit restricted expression to fetal hematopoietic tissue and newborn peripheral blood and appear to be temporally regulated during development. Finally, nucleic acid digestion experiments illustrate that the isoforms appear to be created by an RNA rearrangement event between penta- or octanucleotide direct repeats in adjacent exons. The precise genesis and function of these novel isoforms is unknown at present. Interestingly, each of the four isoforms identified maintain an open reading frame as well as 5' and 3' regulatory regions invoking the possibility that they encode a family of related polypeptides.

Application of fractional mass for the identification of peptide–oligonucleotide cross‐links by mass spectrometry

A method has been developed to identify oligonucleotide-peptide heteroconjugates by accurate mass measurements using MS. The fractional mass (the decimal fraction mass value following the monoisotopic nominal mass) for peptides and oligonucleotides is different due to their differing molecular compositions. This property has been used to develop the general conditions necessary to differentiate peptides and oligonucleotides from oligonucleotide-peptide heteroconjugates. Peptides and oligonucleotides generated by the theoretical digestion of various proteins and nucleic acids were plotted as nominal mass versus fractional mass. Such plots reveal that three nucleotides cross-linked to a peptide produce enough change in the fractional mass to be recognized from non-cross-linked peptides at the same nominal mass. Experimentally, a Cytochrome c digest was spiked with an oligonucleotide-peptide heteroconjugate and conditions for analyzing the sample using liquid chromatography (LC)-MS were optimized. Upon analysis of this mixture, all detected masses were plotted on a fractional mass plot and the heteroconjugate could be readily distinguished from non-cross-linked peptides. The method developed here can be incorporated into a general proteomics-like scheme for identifying protein-nucleic acid cross-links, and this method is equally applicable to characterizing cross-links generated from protein-DNA and protein-RNA complexes.

Quantitative PCR to estimate copepod feeding

Copepods play a central role in marine food webs as grazers of plankton and as key prey for many predators. Therefore, quantifying their specific trophic interactions is critical for understanding the role of copepods in ocean processes. However, because of methodological constraints, it remains difficult to investigate in situ copepod feeding without reliance on laborious intrusive and potentially biased incubation approaches. Recent advances in PCR-based methodologies have demonstrated the feasibility of directly identifying copepod diets based on prey DNA sequences. Yet, obtaining quantitative information from these approaches remains challenging. This study presents results of systematic efforts to develop a quantitative PCR (qPCR) assay targeted to 18S rRNA gene fragments to estimate copepod gut content of specific species of prey algae. These results were first compared to gut content estimates based on fluorescence in the copepod Calanus finmarchicus fed monocultures of two different microalgae species in controlled laboratory studies. In subsequent field studies, we compared feeding rates obtained by microscopy and qPCR for Temora longicornis and Acartia clausi feeding on the haptophyte Phaeocystis globosa in natural blooms. These investigations demonstrate a semi-quantitative relationship between gut content estimates derived from qPCR, gut pigment, and direct microscopy. However, absolute estimates of gut content based on qPCR methodology were consistently lower than expected. This did not appear to be explained by the extraction methods used, or interference by non-target (predator) DNA in the PCR reactions, instead suggesting digestion of prey-specific nucleic acids. Furthermore, the 18S rDNA target gene copy number of the phytoplankton varied with growth phase. Nonetheless, when prey target gene copy number in the ambient water is quantified, the qPCR-approach can be compared to other methods, and then used to semi-quantitatively estimate relative copepod grazing on specific prey in situ without involving further incubations. A distinct advantage of a DNA-based molecular approach compared to gut fluorescence and direct microscopic observation, is the ability to detect non-pigmented and macerated prey. Future studies should aim to correct for breakdown in prey DNA and perform extensive calibrations to other methods in order to achieve a quantitative measure of feeding rates in situ.

The nuclear PP1 interacting protein ZAP3 (ZAP) is a putative nucleoside kinase that complexes with SAM68, CIA, NF110/45, and HNRNP-G

The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.

A Biodegradable, Acellular Xenogeneic Scaffold for Regeneration of the Vocal Fold Lamina Propria

A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.

Ab initio DNA synthesis accelerated by endonuclease

In the presence of an endonuclease that digests double-stranded DNA, DNA polymerase efficiently synthesizes and amplifies DNA from dNTPs in the absence of any added template and primer nucleic acid. This cut-polymerization DNA synthesis (Cut-grow) can be carried out under a wide range of temperature (4-85 degrees C) by many endonucleases and DNA polymerases. The high efficiency of Cut-grow results from an efficient exponential amplification involving digestion-elongation cycles. Our findings suggest that digestion of nucleic acids may play an important role during the evolution of genetic material for procreating the diversification of genetic information on the early earth.

Observations on some unusual cell types in the enigmatic worm Xenoturbella (phylum uncertain)

The inner epithelially organized gastrodermis of the enigmatic simple worms of the genus Xenoturbella contains numerous partly phagocytized cells of two kinds, ciliated cells (PCCs) and muscle cells (PMCs). PCCs and PMCs have features of undifferentiated cells and do not derive from differentiated adult cells. Homology of phagocytized cells to pulsatile bodies in acoel and nemertodermatid flatworms is therefore rejected. The phagocytized cells might represent an hitherto unknown process of regeneration in Xenoturbella. The phagocytized material contains as much DNA as in all mitochondria and nuclei of the living cells. This is probably caused by lack of digestion of nucleic acids. The genome size of Xenoturbella bocki was determined. It has a C-value of about 0.55 pg.

Development of an Esophagus Acellular Matrix Tissue Scaffold

A cell-extraction protocol yielding an esophagus acellular matrix (EAM) scaffold for use in tissue engineering of an esophagus, including hypotonic lysis, multiple detergent cell extraction steps, and nucleic acid digestion, was developed in a rat model. Histological techniques, burst pressure studies, in vitro esophageal epithelial cell seeding, and in vivo implantation were used to assess cell extraction, extracellular matrix (ECM) preservation, and biocompatibility. Microscopy demonstrated that cell extraction protocols using sodium dodecyl sulfate (SDS) (0.5%, wt/vol) as a detergent resulted in cell-free EAM with retained ECM protein collagen, elastin, laminin, and fibronectin. Burst pressure studies indicated a loss of tensile strength in EAMs, but at intraluminal pressures that were unlikely to affect in vivo application. In vitro cell seeding studies exhibited epithelial cell proliferation with stratification similar to native esophagi after 11 days, and subcutaneously implanted EAMs displayed neovascularization and a minimal inflammatory response after 30 days of implantation. This study presents an esophagus acellular matrix tissue scaffold with preserved ECM proteins, biomechanical properties, and the ability to support esophageal cell proliferation to serve as the foundation for a tissue-engineered esophagus.

A surface-mediated origin of the RNA world: biogenic activities of clay-adsorbed RNA molecules

The involvement of clay surfaces in the origin of the first genetic molecules on Earth has long been suggested. However, the formation of these polymers was not sufficient by itself to initiate the evolutionary process leading to the appearance of life. These macromolecules had to persist in primeval habitats so that their biological potentiality could be expressed. In this study, we assess the possibility of development of the RNA world on a clay substrate by investigating the capacity of different RNA molecules adsorbed/bound on the clay minerals montmorillonite (M) and kaolinite (K) to persist in the presence of a degrading agent (RNase-A), to interact specifically with complementary RNA strands, and to transmit the information contained in their nucleotide sequences. The RNase-A degradation of clay-adsorbed 23S rRNA from Escherichia coli was significantly slower (75–80%) than that observed for free rRNA, and the complete digestion of nucleic acid in the presence of clay was obtained in 2 vs. 1 h. Clay-adsorbed Poly[A] homopolymer was able to recognize the complementary Poly[U] homopolymer present in the surrounding water solution and to establish a specific interaction (association) with it, possibly leading to the formation of double strands. Reverse transcription and amplification (RT–PCR) amplification of free and clay-adsorbed 16S indicated that the presence of clay particles partially reduced the efficiency and processivity of reverse transcriptase but did not inhibit its activity, demonstrating that clay-adsorbed RNA is still available for enzymatic replication. These findings indicate that primordial genetic molecules adsorbed on clay minerals would have been protected against degrading agents present in the environment and would have been in the right conditions to undergo evolutionary processes.

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67.

To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.

Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications.

Vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped replication-defective retroviral particles are pantropic and amenable to concentration to high titer by ultracentrifugation. These features have allowed development of effective retroviral transduction protocols for stem cells in vitro as well as for tissue engineering in vivo. However, retroparticle ultracentrifugation protocols will also copellet cellular and subcellular debris released from retroviral producer cell lines during vector manufacture. We have analyzed concentrated vector preparations by chromatography and have found that a significant amount of genomic DNA released from producer cells coconcentrates with retroviral particles. In an effort to generate high-purity retroparticle preparations, devoid of subcellular contaminants and contaminating genomic DNA, we have developed a process using size-exclusion chromatography combined with host cell nucleic acid digestion and concentration by ultrafiltration. The procedure allowed for a final recovery of 19 +/- 0.4% infectious viral particles from unfractionated starting material, with an average retroparticle concentration of 7.7 x 10(7) +/- 1.5 x 10(6)/ml. The intact virus is of high purity, >90% as determined by anion-exchange high-performance liquid chromatography. Retroparticle structure appeared intact as determined by negative stain electron microscopy and purified virus was functional and allowed for efficient transduction of primary human bone marrow stromal cells in vitro. In conclusion, we have developed a VSV-G retrovector purification process that can be applied to large-scale retroviral production ideal for cell and gene therapy applications.

A Safety Evaluation of Genetically Modified Feedstuffs for Livestock Production; the Fate of Transgenic DNA and Proteins

Two genetic constructs used to confer improved agronomic characteristics, namely herbicide tolerance (HT) in maize and soyabean and insect resistance (Bt) in maize, are considered in respect of feeding to farm livestock, animal performance and the nutritional value and safety of animal products. A review of nucleic acid (DNA) and protein digestion in farm livestock concludes that the frequency of intact transgenic DNA and proteins of GM and non-GM crops being absorbed is minimal/non existent, although there is some evidence of the presence of short fragments of rubisco DNA of non-GM soya in animal tissues. It has been established that feed processing (especially heat) prior to feeding causes significant disruption of plant DNA. Studies with ruminant and non-ruminant farm livestock offered GM feeds demonstrated that animal performance and product composition are unaffected and that there is no evidence of transgenic DNA or proteins of current GM in the products of animals consuming such feeds. On this evidence, current HT and Bt constructs represent no threat to the health of animals, or humans consuming the products of such animals. However as new GM constructs become available it will be necessary to subject these to rigorous evaluation.

Nucleosides Are Efficiently Absorbed by Na+-Dependent Transport Across the Intestinal Brush Border Membrane in Veal Calves

In previous work, a comparatively high capacity for Na+-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was observed in dairy cows, which might be related to digestion of the large amount of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na+-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amounts of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na+-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concentration gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids observed by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids associated with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.

Characteristics of Na(+)-dependent intestinal nucleoside transport in the pig.

Since the capacity of nucleic acid digestion and absorption appears to be comparatively high in the pig, we investigated the properties of transport of (3)H-labelled nucleosides across the porcine intestinal brush border membrane (BBM) using BBM vesicles isolated from the small intestine of slaughter pigs. In the presence of a transmembrane Na(+) gradient, uridine, thymidine and guanosine transiently accumulated in the vesicular lumen beyond the equilibrium (60 min) value suggesting the presence of Na(+)/nucleoside cotransporters in the BBM. The findings of inhibitory studies are consistent with the presence of two Na(+)-dependent nucleoside transporters with overlapping substrate specificity, one for pyrimidine nucleosides (N2) and one for purine nucleosides (N1). Guanosine appeared to be a specific substrate for N1, while this applies to thymidine for N2. Transport of thymidine and guanosine were also inhibited by 2 mmol/l D-glucose and alpha-methyl-D-glucoside. The maximal transport capacity (V(max)) for Na(+)-dependent thymidine and guanosine transport were much higher than reported for other monogastric species. Unlike in other species tested, there was no proximal-to-distal gradient, neither in nucleoside transport activity nor in the inhibition of nucleoside transport by monosaccharides in the porcine small intestine. The high intestinal nucleoside transport activity may contribute to the high digestive capacity for nucleic acids in the pig.

Properties of Na(+)-dependent nucleoside transport in the proximal and distal small intestine of cows.

Large amounts of nucleic acids associated with rumen microorganisms are digested in the proximal part of the small intestine of ruminants. We studied how the proximal-distal gradient in nucleic acid digestion is related to activity of Na(+)-nucleoside transporters in brush border membrane vesicles isolated from the proximal and distal small intestine of cows. Two Na(+)-dependent nucleoside transporters with overlapping substrate specificity were shown to be present at the two intestinal sites, one for pyrimidine nucleosides and one for purine nucleosides. Affinity constants (K(m)-values) for both thymidine and guanosine transport were similar at the two intestinal sites, while transport capacity (V(max)) was 2-3 times higher in the proximal than in the distal small intestine. Glucose and alpha-methyl-D-glucoside (0.1 mmol/l or 2 mmol/l) inhibited transport of thymidine and guanosine markedly only in the proximal small intestine. It is concluded that absorption of nucleosides by the two Na(+)-nucleoside transporters reflects the proximal-distal gradient in nucleic acid digestion.

Molecular Biology and Immunology for Clinicians

Electrophoresis is a powerful tool used for the separation of proteins and nucleic acids by their size. For the latter, often very large molecules, separation techniques typically use agarose gels rather than the polyacrylamide gels used to separate proteins. Once separated and transferred to a membrane support, one can detect nucleotides by identifying unique sequences of their purine and pyrimidine bases using a complementary DNA probe specially synthesized to be specific for the nucleic acid of interest. One can electrophoretically separate and detect DNA (Southern blot analysis) or RNA (Northern blot analysis). These techniques depend on a DNA probe hybridizing with the target DNA immobilized on a membrane. Hybridization techniques are sensitive enough to detect differences (caused by either mutation or inherited differences) in the sequences of genetic material using a technique based on the digestion of nucleic acids by restriction endonucleases, which produce restriction fragment-length polymorphisms. DNA probe hybridization can be also used to detect genetic material within cells in in situ hybridization.

Application of high-performance liquid chromatography for recognition of covalent nucleic acid modification with anticancer drugs

Covalent modification of DNA by antineoplastic agents represents a potent biochemical lesion which can play a major role in drug mechanism of action. The ability to measure levels of DNA covalent modifications in target cells in vivo may, therefore, be seen as the ultimate form of therapeutic drug monitoring. Additionally, elucidation of the structure of critical DNA adducts and definition of their role in tumour cell cytotoxicity will provide more selective targets for rational drug design of new cancer chemotherapeutic agents. High-performance liquid chromatography has contributed significantly to all these areas. In vivo levels of nucleic acid covalent modifications are in the range of 1 in 10 5–10 8 nucleotides precluding the use of conventional high-performance liquid chromatographic detection methods. Several classes of natural product anticancer drugs have been shown to bond covalently to nucleic acids under optimal laboratory conditions. These have proved more accessible to high-performance liquid chromatographic analysis because of their lipophilicity and strong UV chromophores. However, the majority of experimental evidence to date suggests that with the exception of mitomycin C and morpholino-anthracyclines these compounds do not exert their primary mechanism of action through nucleic acid covalent modification. DNA adducts of alkylating and platinating agents are more difficult to detect by high-performance liquid chromatography and can be chemically unstable. These compounds interact with DNA on the basis of chemical kinetics. Thus, the principle sites of attachment tend to be with the most nucleophilic base (guanine) at its most reactive centre (N-7 position). Limited in vivo high-performance liquid chromatographic studies with all classes of anticancer drugs indicate a much more complex pattern of adductation than would have been anticipated from in vitro studies alone. Some of these differences are probably due to methodological artefacts but these studies stress the need for sensitive detection methods and reliable sample preparation (nucleic acid extraction and digestion techniques) when attempting to determine nucleic acid covalent modifications by anticancer drugs.

Isoelectric focusing and 2D electrophoresis of the human androgen receptor

Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polycrylamide gels before and after photoaffinity labeling with [ 3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 ± 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated IEF sample buffer containing no salts, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 ± 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with M w 90–95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with M w 90–95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc +) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and M w 90–95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc +) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.

Detection of single-stranded DNA in scrapie-infected brain by electron microscopy

The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 × 10 6 daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains.

Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides

A volatile buffer has been adapted for use with ionexchange high-performance liquid chromatography in the analysis and preparation of oligonucleotides. The system employs a commercial weakly basic anion-exchange column containing a DEAE-derivatized silica gel and eluted with a volatile buffer gradient of triethylamine acetate and acetonitrile. Nucleic acid digests and oligonucleotides synthesized by chemical or enzymatic methods can be analyzed or purified with nearly quantitative recovery following solvent volatilization.