Mitsutomo Tsuhako

High-resolution separation of DNA restriction fragments by capillary electrophoresis in cellulose derivative solutions

The performance and the efficiency of several cellulose derivatives as a molecular sieving agent for the capillary electrophoretic separation of DNA restriction fragments were investigated. All fragments up to 12,000 base pairs (bp) in the 1-kbp DNA ladder were resolved using linear polyacrylamide-coated capillaries filled with a buffer solution containing 0.5% cellulose derivative and the separation was completed within 17 min. High-concentration (0.7%) cellulose derivative solutions are effective for the complete separation of small fragments (50-1000 bp) of a HincII and a HaeIII digest of phi X174 DNA. A plate number of 0.5-1 x 10(6) plates per metre was achieved. The migration time and the resolution of DNA fragments were manipulated by varying several parameters, such as the size (viscosity) and the concentration of cellulose derivatives and the applied field strength. Some guidelines are presented for choosing these parameters, depending on the size of the DNA fragments being separated.

Gel-filled capillaries for nucleic acid separations in capillary electrophoresis

Abstract Capillary gel electrophoresis is an exciting new technique for ultrahigh resolution of nucleic acids. Its primary practical barrier is the difficulty of producing suitable gel-filled capillaries. In this review, recent progress in the methods to overcome the difficulty is discussed. The great potential of capillary gel electrophoresis for the human genome investigations is illustrated.

Structural study of phosphate groups in layered metal phosphates byhigh-resolution solid-state31P NMR spectroscopy

In studies of a series of crystals of layered metal phosphate compounds by magic angle spinning (MAS) NMR spectroscopy, it has been found that there is a distinct correlation between the 31 P isotropic chemical shift value and the type of phosphate group. The isotropic 31 P chemical shifts of (H 2 PO 4 ) - , (HPO 4 ) 2- and (PO 4 ) 3- groups appear around δ -10, -20 and -30, respectively. These assignments were made unambiguously by measuring the 31 P MAS NMR spectra with and without 1 H high-power decoupling together with the cross-polarization transfer technique between 1 H and 31 P nuclei (CP MAS spectra).

Adsorption of formaldehyde by polyamine-intercalated α-zirconium phosphate

Gaseous formaldehyde adsorption mechanism of diethylenetriamine- and pentaethylenehexamine-intercalated α-zirconium phosphate was demonstrated to be self oxidation-reduction of formaldehyde, that is, Cannizzaro reaction, in the interlayer space as evidenced by XRD and solid-state NMR. This fact suggests that these intercalation compounds can be used as a reaction field of self oxidation-reduction of formaldehyde, and also as adsorbents of formaldehyde, which causes sick-house syndrome.

Comparison of high-performance liquid chromatography with capillary gel electrophoresis in single-base resolution of polynucleotides

Abstract High-resolution separations of polynucleotides were performed using capillary gel electrophoresis (cGE) and high-performance liquid chromatography (HPLC) with reversed-phase, ion-exchange and mixed-mode columns. Electropherograms showing cGE separations of single-stranded homopolynucleotides were presented and compared with HPLC separations according to the chain length of the polynucleotides. The resolving power of cGE is much higher than that of any HPLC mode. The chain length limits for complete separation within 60 min by cGE, reversed-phase, ion-exchange and mixed-mode HPLC are ca. 250, 30, 40 and 40 nucleotides, respectively. The plate number which is achieved for cGE of 3 · 106-7 · 106 plates/m is about ten times larger than those of all HPLC modes of 4 · 104-8 · 105 plates/m. The reproducibility of the migration time in cGE (2–4%) is comparable to those of retention times in HPLC in several separation modes (1–3%).

Temperature-programmed capillary affinity gel electrophoresis for the sensitive base-specific separation of oligodeoxynucleotides

Abstract High-performance base-specific separations of oligodeoxynucleotides were performed by temperature-programmed capillary affinity gel electrophoresis (CAGE), in which poly(9-vinylacenine) (PVAd) was utilized as an affinity ligand. The migration behaviour of oligodeoxynucleotides was investigated at different capillary temperatures. The migration time and resolution of oligodeoxyadenylic acids, which do not interact with PVAd, decrease with increase in temperature as in capillary gel electrophoretic separation. The migration behaviour of oligothymidylic acids, which interact with PVAd, is manipulated by varying the capillary temperature, which leads to changes in the dissociation process of specific hydrogen bonding between oligothymidylic acids and PVAd. The implementation of temperature-programmed CAGE was illustrated by the selective and sensitive base recognition of oligodeoxynucleotides with efficiencies as high as several times 106 plates per metre.

Comparative study of non-porous anion-exchange chromatography, capillary gel electrophoresis and capillary electrophoresis in polymer solutions in the separation of DNA restriction fragments

Abstract HPLC using a 100-mm column packed with a non-porous anion exchanger gave high-resolution separations of DNA restriction fragments up to 8000 base pairs (bp). High-resolution separation of DNA restriction fragments up to 12 000 bp was achieved using capillary gel electrophoresis (CGE) and capillary electrophoresis (CE) in entangled polymer solutions. These methods were compared with respect to their performance and efficiencies in the resolution of DNA restriction fragments. The resolving power of CGE is higher than those of the other techniques. The 50–100 bp resolution of DNA fragments up to 500 bp was realized using HPLC, 50 bp resolution using CE in entangled polymer solutions and 10 bp resolution using CGE. The plate number that was achieved with CGE of 3 · 106 plates/m was higher than those of HPLC of 1 · 106 and CE in polymer solutions of 7 · 105 plates/m. The advantages and limitations of HPLC and capillary electrophoretic techniques are discussed.

Enhanced separation of DNA sequencing products by capillary electrophoresis using a stepwise gradient of electric field strength.

The effect of the electric field strength gradient on the separation of DNA sequencing fragments was investigated. We demonstrate that the stepwise gradient of electric field improves the separation of DNA sequencing fragments more than 500 bases in size and diminishes the analysis time for DNA sequencing of lager DNA fragments. The use of the electric field strength gradient induces an increase in the theoretical plate number as predicted by the theoretical formulation discussed in this paper.

Unusual adsorption mechanism for carboxylic acid gases by polyamine-intercalated α-zirconium phosphate

The adsorption mechanism of gaseous carboxylic acid by diethylenetriamine- or pentaethylenehexamine-intercalated α-zirconium phosphate was examined in detail using XRD and solid-state 13C and 31P NMR. The adsorption of acetic acid, propanoic acid, and butyric acid resulted in the co-intercalation of the carboxylic acid molecules into polyamine-intercalated α-zirconium phosphate to expand their interlayer distance by 0.64–0.97 nm. In the case of formic acid, its adsorption induced the rearrangement of the polyamine molecules within the interlayer space.

A one-step phosphorylation of d-aldohexoses and d-aldopentoses with inorganic cyclo-triphosphate

The phosphorylation reaction by inorganic cyclo-triphosphate (P3m) having a six-membered ring was examined for D-aldohexoses and D-aldopentoses in aqueous solution. Similar to the process for D-glucose, D-galactose, D-xylose or D-allose were phosphorylated with P3m to give stereoselectively beta-D-galactopyranosyl 1-triphosphate, beta-D-xylopyranosyl 1-triphosphate or beta-D-allopyranosyl 1-triphosphate with maximum yields of 31.3, 32.5 or 32.1%, respectively. On the other hand, in the reaction of D-ribose, D-lyxose, D-mannose or D-arabinose with P3m, the yields of beta-D-ribopyranosyl 1-triphosphate, alpha-D-lyxopyranosyl 1-triphosphate, alpha-D-mannopyranosyl 1-triphosphate or alpha-D-arabinopyranosyl 1-triphosphate were 8.0, 16.5, 9.6 or 14.1%, respectively. The phosphorylation mechanism of D-aldopyranoses with P3m was also discussed.