Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Infrared spectroscopy was used to identify hydration-sensitive structural differences between single- stranded phosphorothioate (PS) and phosphodiester (PO) oligodeoxyribonucleotides. Spectra were recorded in the mid-infrared region, 500-1800 cm-1, at relative humidities between 0 and 98%; the PS and PO spectra are substantially different. The hydration effects on spectral bands in these single-stranded oligodeoxyribonucleotides is markedly different from such behavior in double- and triple-stranded oligodeoxyribonucleotides. A strong absorption occurs at 656 cm-1 in the phosphorothioate sample which is completely absent from the PO spectra. Gravimetric measurements were carried out on one PS and one PO sample to monitor and confirm hydration. The calculated BET adsorption constants [Brunauer, S., Emmett, RH. and Teller, E. (1938) J. Am. Chem. Soc., 60, 309-319] are 1.2 and 1.4 water molecules per nucleotide in the first hydration layer of PS and PO respectively. While the gravimetric data indicate that the single-stranded oligodeoxyribonucleotides hydrate very similarly to duplex DNA, the mid-infrared conformational marker bands are strikingly different from those observed for duplex DNA. In particular, the Vas of the phosphate group (PO2) at 1222 cm-1 in the single-stranded PO spectra is independent of relative humidity.     

Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase

Despite its unique ability to ligate single-stranded DNA molecules, T4 RNA ligase has so far seen little use in molecular biology due to long reaction times, modest yields, and apparent inability to promote ligation of long oligodeoxyribonucleotides. We describe here a set of reaction conditions which dramatically shorten the reaction time and give reproducible 40 to 60% ligation of DNA fragments of up to 40 bases in length. These improvements open promising new fields of application to T4 RNA ligase.     

Interaction of the RecA protein of Escherichia coli with single-stranded oligodeoxyribonucleotides.

The RecA protein of Escherichia coli performs a number of ATP-dependent, in vitro reactions and is a DNA-dependent ATPase. Small oligodeoxyribonucleotides were used as DNA cofactors in a kinetic analysis of the ATPase reaction. Polymers of deoxythymidilic acid as well as oligonucleotides of mixed base composition stimulated the RecA ATPase activity in a length-dependent fashion. Both the initial rate and the extent of the reaction were affected by chain length. Full activity was seen with chain lengths > or = 30 nt. Partial activity was seen with chain lengths of 15-30 nt. The lower activity of shorter oligonucleotides was not simply due to a reduced affinity for DNA, since effects of chain length on KmATP and the Hill coefficient for ATP hydrolysis were also observed. The results also suggested that single-stranded DNA secondary structure frequently affects the ATPase activity of RecA protein with oligodeoxyribonucleotides.     

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones

A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol–chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2 min instead of 24 h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8 ± 0.2 °C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40 ng/μl and proved to be a superior alternative to the phenol–chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.     

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides

RecBCD is an ATP-dependent helicase and exonuclease which generates 3′ single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5′-GCTGGTGG-3′) in double-stranded DNA (ds DNA), an event critical to the generation of the 3′-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Chi⁺) or a single base change that abolishes the Chi sequence (Chi^o), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi⁺ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi^o oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.     

The use of a monolithic column to improve the simultaneous determination of four cephalosporin antibiotics in pharmaceuticals and body fluids by HPLC after solid phase extraction--a comparison with a conventional reversed-phase silica-based column

The use of a monolithic column (Chromolith, SpeedROD RP-18e, by Merck) was studied on the determination of cephalosporin antibiotics. Results were compared with those from a previously developed analytical method using conventional silica-based analytical column. A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins: Cephalexine and Cephadroxil (first generation), Cefaclor (second generation) and Cefotaxim (third generation) in pharmaceuticals as well as in human blood serum and urine. Hydroflumethiazide (HFM) (3,4-dihydro-6(trifluoromethyl)-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide) was used as an internal standard at a concentration of 1.5 ng/microL. A rectilinear relationship was observed up to 5 ng/microL for the four compounds. Analysis time was less than 4 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=8) and was found to be satisfactory with high accuracy and precision results. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked serum samples was in the range from 88.7 to 107.8%, while for urine samples recovery was from 98.0 to 105.6%. By comparing the figures of merit for the monolithic column and the silica-based one, regarding the determination of the four cephalosporins investigated in the present study, the outstanding efficiency of the monolithic column can be noticed.     

A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.     

A new method for sequence analysis of oligodeoxyribonucleotides.

The structure of an oligodeoxyribonucleotide may be determined by a simple two-dimensional separation on a polyethyleneimine-cellulose thin layer sheet. Chromatography in the first dimension fractionates by chain length a nested set of fragments that are generated by subjecting the oligomer to partial spleen phosphodiesterase degradation and then labelling their non-common ends with 32P using polynucleotide kinase. A subsequent in situ treatment with nuclease Bal 31 produces labelled mononucleotides, and these are identified by chromatography in the second dimension. Since the method does not identify the 3' terminal nucleotide, a convenient procedure involving 3' end labelling followed by enzymatic digestion to monomers has been developed for this purpose. This approach to sequence analysis also has the advantage of permitting assignment of the identity and location of any modified or unusual bases within the oligonucleotide.     

Three-minute high-performance liquid chromatographic assay for NMN adenylyltransferase using a 20-mm-long reversed-phase column

NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly catalyzes the synthesis of NAD from ATP and NMN. In this paper, we describe a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C₁₈ reversed-phase (RP) column. The activity was measured by separating in less than 3 min the substrates (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22°C. NAD was directly quantitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfully applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) cells.     

Determination of cephalosporins in biological material by reversed-phase liquid column chromatography

Seven cephalosporins (β-lactam antibiotics), viz. cefazolin, cephalotin, cefoxitin, cefotaxime, cefamandole, cefuroxime and cefoperazone (T 1551) were determined in biological material. The compounds were extracted from acid-treated body fluids into chloroform—1-pentanol (3:1) and re-extracted into a small volume of an aqueous phase at pH 7, which was injected into the chromatographic column. The chromatographic support was μBondapak C_1a (10 μm) and the mobile phase was a mixture of 0.01 M acetate buffer (pH 4.8) and methanol or acetonitrile. Detection limits are about 50 ng/ml for extractions from 1 ml of serum and have permitted pharmacokinetic studies of the seven cephalosporins.     

Determination of isofezolac in biological fluids by reversed-phase liquid column chromatography

A rapid, sensitive, and specific reversed-phase high-performance liquid chromatography assay was developed for the determination of 1,3,4-triphenylpyrazole-5-acetic acid (isofezolac) in plasma and urine. The assay involves extraction into diethyl ether from plasma buffered at pH 4.4. The organic phase is evaporated and the residue, dissolved in the mobile phase [acetonitrile—water—0.2 M phosphate buffer (pH 3) (65 : 15 : 20)] is chromatographed at a flow-rate of 1.5 ml/min. The drug is detected by its UV absorption (detection limit 100 ng/ml) or its very intense fluorescence (detection limit 10 ng/ml). Absolute analytical recoveries for isofezolac varied from 92.9 to 100.4%. The accuracy is ca. 1%. Each separation requires about 6 min. This method was applied successfully to the determination of isofezolac in humans for pharmacokinetic studies.     

Analysis of endo-beta-N-acetylglucosaminidase activity by high-pressure liquid chromatography on a silica-based chemically bonded octadecyl column

We have devised a new method for assaying the endo-β-N-acetylglucosaminidase activity by using the dansyl asparaginyl oligosaccharide, (Man)₅(GlcNAc)₂-Asn-DNS, as the substrate and analyzing the product, GlcNAc-Asn-DNS, by a reverse-phase high-pressure liquid chromatography using a silica-based chemically bonded octadecyl column (Waters μBondapack C₁₈). The column is eluted with 8% acetonitrile in 25 mm sodium borate buffer, pH 7.5, at 3 ml/min. The effluent is monitored by a Perkin-Elmer LC-75 uv monitor at 213 nm and a Perkin-Elmer LC-1000 fluorescence monitor (excitation, 313 nm; emission, 540 nm). Under these conditions, GlcNAc-Asn-DNS is well separated from (Man)₅(GlcNAc)₂-Asn-DNS and the analysis can be completed in 5 min. The peak height is used to quantify the dansyl derivatives. Under the conditions described above, the lower limit of detection is 0.1 nmol of dansyl glycopeptides.     

Comparison of ion-pair and amide-based column reversed-phase liquid chromatography for the separation of thiamine-related compounds

Two reversed-phase chromatographic methods for the separation of thiamine and related compounds are compared. The first procedure is based on the ion-pair technique using an octadecylsilica column, while the second uses a new amide-based stationary phase, which avoids the need to form ion-pairs, leading to narrower peaks and a simpler mobile phase. Analyses were performed by gradient elution and a photo-diode array was used for detection. Specificity was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity index with commercial standards. The procedures were applied to the determination of thiamine-related compounds in pharmaceutical preparations and urine. No preliminary sample treatment was required.     

A new linkage for solid phase synthesis of oligodeoxyribonucleotides.

An aryl diisocyanate has been used to attach an appropriately protected 2'-deoxyribonucleoside bearing a free 3'-hydroxyl group, to a long chain alkylamine controlled pore glass support via a urethane moiety, in a simple two step procedure. This obviates the need for the preparation and short column chromatographic purification of the 2'-deoxyribonucleoside-3'-O-succinates required for preparation of the widely used succinyl linked supports. The greater stability of the urethane bond compared to an ester bond led to substantially higher yields of oligodeoxyribonucleotides prepared by the solid phase phosphotriester method. More than twenty oligodeoxyribonucleotides have already been synthesized on the glass support bearing the new linkage.     

Effective selective modification of a single-stranded fragment of DNA with alkylating derivatives of short oligodeoxyribonucleotides in the presence of reaction effectors N-(2-hydroxyethyl)phenazine derivatives of oligodeoxyribonucleotides

Tri-, tetra-, penta- and hexanucleotides bearing a reactive 4-(N-methylamino-N-2-chloroethyl)benzylamide group can effectively and selectively modify a single-stranded DNA fragment (302 nucleotides) in the presence of effectors, N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides complementary to DNA sequences adjacent to the binding site of the reagent. The reagents investigated modify not only single-stranded but also secondary-structured DNA regions. The modification extent depends on the length of oligonucleotide parts of the reagent and effector. A gap between the two stretches associated with the target DNA prevents the effector from functioning. The substitution of an octanucleotide effector by two tetranucleotide ones only slightly reduces the modification extent with a hexanucleotide reagent. A very efficient and specific modification can be achieved by using two effectors flanking the reactive oligonucleotide derivative. The approach leads to the modification extent of up to 89% with a hexanucleotide reagent.     

A novel method for preparing single-stranded DNA for pyrosequencing

Pyrosequencing technology is a powerful genotyping tool that requires the generation of single stranded DNA. Currently, two simple, solid-phase-based methods are available for this, but they require special equipment, they are not automated, and they are relatively expensive because of the need for biotinylated polymerase chain reaction primers. In this article, an enzymatic liquid-phase method for the generation of high-quality, single-stranded DNA, and its novel use for Pyrosequencing are described. The method has also been fully automated.