Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography

This work describes the application of the fully automated high-performance liquid chromatographic system to the analysis of PCR-amplified products. Efficient separations of both DNA restriction fragments and PCR products were performed using an anion-exchange DEAE-NPR column, packed with 2.5-μm nonporous particles. The automated HPLC method was employed for the separation, quantitation, and purification of PCR products in less than 10 min in a single step.     

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO₄^?, subjected to preliminary purification on a Cu²⁺-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.     

Separation of DNA fragments and single strand conformation polymorphism analysis in bare capillaries using poly(acrylamide–dimethylacrylamide) as a separation medium

A short chain poly(acrylamide–dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA–capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis.     

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 μm). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.     

Effect of temperature on the separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis: a comparative study

This study investigates the effect of experimental temperature on the separation of DNA fragments, 21–587 bp, by both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The results show that the temperature plays an important role in the HPLC separation of DNA fragments. The optimum temperature was found to be between 40 and 50°C for HPLC, while 25°C was the optimum temperature for the CE separation. Also, although CE migration times became shorter, efficiency and resolution decreased with an increase in temperature from 25 to 50°C, but the separation was not significantly affected. Also, the optimum HPLC temperature might be different depending on the fragment sizes to be resolved.     

Analysis of polymerase chain reaction products by high-performance liquid chromatography with fluorimetric detection and its application to DNA diagnosis

We describe the development of a sensitive high-performance liquid chromatographic (HPLC) method for polymerase chain reaction (PCR) products using bisbenzimide (Hoechst 33258 dye) based fluorimetric detection. The detection limit and specificity for double-strand DNA detection are improved in comparison with HPLC with UV absorbance detection. This HPLC, using a column packed with diethylaminoethyl-bonded non-porous resin particles, was applied to the detection of allele-specific PCR and restriction fragment length polymorphism analysis. We also developed a hybridization method analyzed by HPLC. DNA fragments (149 bp) containing the mutation site (C→A,G,T) in the N-ras gene were amplified by PCR. Fluorescein isothiocyanate (FITC)-labeled DNA probes were also prepared by PCR using FITC-labeled 5′ primer. Analysis of mutation was performed by the separation of a hybrid and non-reactive DNA probe with HPLC with fluorimetric detection after the hybridization of target DNA (149 bp) and a FITC DNA probe. The effects of various factors on hybridization were examined to establish optimal assay conditions. Under the conditions determined, a point mutation in PCR products obtained from the N-ras gene could be detected specifically by this method. The analysis of PCR products by HPLC may potentially be useful for DNA diagnosis.     

Separation of DNA fragments by high-resolution ion-exchange chromatography on a nonporous QA column

A nonporous QA column (a strong anion exchanger) was used for HPLC of DNA fragments. This column was successfully employed to separate small (ca. 10 bp) and intermediate size (ca. 10 kbp) DNA fragments from each other. The column also separated double-stranded DNA from its single-stranded form, and circular DNA molecules from linear ones. The entire separation process was completed within 60 min. The recovery of DNA fragments in each run was above 95%. High resolution was obtained both at an analytical level (microgram scale) and at a preparative level (100 micrograms scale). In view of time efficiency, recovery, and resolution, the nonporous QA column is superior to other porous ion-exchange columns and expected to be a very useful tool in molecular biological studies.     

High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

Rapid analysis and purification of polymerase chain reaction products by high-performance liquid chromatography

This report describes the use of high-performance liquid chromatography (HPLC) for the rapid analysis and purification of the polymerase chain reaction products. Employing a new anion-exchange nonporous column, efficient separations of both DNA restriction fragments and amplified PCR products are achieved in 10 to 20 minutes and quantitated within +/- 10%. The performance of the HPLC technique is described in terms of resolution, reproductibility, sensitivity and micropreparative capability and compared to that of gel electrophoresis for this application.     

Two-dimensional high-performance liquid chromatographic method for assaying S-adenosyl-l-methionine and its related metabolites in tissues

S-Adenosyl-l-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decar☐ylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0–10% acetonitrile gradient in 10 m M ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 μm, 250 mm × 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15–400 m M), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30–500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9–22% for rat liver and 6–24% for mast cells.     

Size-dependent, chromatographic separation of double-stranded DNA which is not based on gel permeation mode

A new procedure for size-dependent fractionation of DNA was investigated. DNA fragments ranging from 10 to 40 kbp were separated by using columns for high-performance gel permeation chromatography. However, the order of elution was opposite to that which would be expected for gel permeation chromatography, i.e., smaller fragments were eluted faster than larger fragments, though separation based on normal gel permeation chromatography was observed when smaller DNA fragments (less than 5 kbp) were applied. The size range of DNA which can be resolved by this new procedure was found to depend on both particle size and flow rate; the use of a column packed with smaller particles or the application of a faster flow rate enabled us to resolve smaller DNA fragments, but the pore size or chemical nature of the column packing had scarcely any effect on the resolution. This mode of separation was attained by using both silica and polymer packings. The results suggest that the separation is based on a hydrodynamic phenomenon.     

Monolithic capillary columns for liquid chromatography-electrospray ionization mass spectrometry in proteomic and genomic research

Peptides, proteins, single-stranded oligonucleotides, and double-stranded DNA fragments were separated with high resolution in micropellicular, monolithic capillary columns prepared by in situ radical copolymerization of styrene and divinylbenzene. Miniaturized chromatography both in the reversed-phase and the ion-pair reversed-phase mode could be realized in the same capillary column because of the nonpolar character of the poly-(styrene/divinylbenzene) stationary phase. The high chromatographic performance of the monolithic stationary phase facilitated the generation of peak capacities for the biopolymers in the range of 50-140 within 10 min under gradient elution conditions. Employing volatile mobile phase components, separations in the two chromatographic separation modes were on-line hyphenated to electrospray ionization (tandem) mass spectrometry, which yielded intact accurate molecular masses as well as sequence information derived from collision-induced fragmentation. The inaccuracy of mass determination in a quadrupole ion trap mass spectrometer was in the range of 0.01-0.02% for proteins up to a molecular mass of 20000, and 0.02-0.12% for DNA fragments up to a molecular mass of 310000. High-performance liquid chromatography-electrospray ionization mass spectrometry utilizing monolithic capillary columns was applied to the identification of proteins by peptide mass fingerprinting, tandem mass spectrometric sequencing, or intact molecular mass determination, as well as to the accurate sizing of double-stranded DNA fragments ranging in size from 50 to 500 base pairs, and to the detection of sequence variations in DNA fragments amplified by the polymerase chain reaction.     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE–glucomannan gel, and direct CL detection of HPOs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100, which could solubilize lipids, was essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE–glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.     

Analysis of free nucleotide pools of mouse liver tissue by high-pressure liquid chromatography (HPLC)

In this paper, analysis of free nucleotides from mouseliver tissue during different day times has been described. Perchloric acid extract of mouse liver tissue was neutralized with tri-N-octylamine in trichlorotriflouroethane and after removal of ClO4(-), subjected to preliminary purification on a Cu(2+)-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure used in the study gave a good resolution of free nucleotides on a single column.     

Rapid analysis and quantitation of PCR products by high-performance liquid chromatography

HPLC utilizing a high efficiency anion-exchange column provides a rapid and easily automated technique for the qualitative and quantitative analysis of subnanogram to microgram amounts of DNA fragments generated by the PCR. The accuracy, precision and linearity of this method exceed those attainable with electrophoretic techniques. In addition, because of the nondestructive nature of HPLC, the desired PCR amplification product can be purified for subsequent utilization. Thus, liquid chromatography extends the utility of the PCR technique to those applications requiring precise quantitation.     

High resolution of multiple forms of red blood cell enzymes using a Toyopearl DEAE 650 S.

We have investigated a new anion exchange chromatographic support (Toyopearl DEAE 650 S) which simultaneously allows easily to remove hemoglobin from hemolysates and to obtain a very high resolution of enzymes present in multiple forms. The results obtained are better than those obtainable using an anion-exchange HPLC column. The data obtained at analytical level suggest a wider use of this new matrix also for preparative purposes without significant changes in the resolution.     

Analytical biochemistry of DNA–protein assemblies from crude cell extracts

Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.     

Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.