Base stacking: pH-mediated on-column sample concentration for capillary DNA sequencing.

Y Xiong; S R Park; H Swerdlow

doi:10.1021/ac980376j

Home/Archive/PubMed

PubMed · 9737211

Base stacking: pH-mediated on-column sample concentration for capillary DNA sequencing.

Y Xiong·S R Park·H Swerdlow

View original source ↗Export metadata ↓

Abstract

An on-column sample concentration method for capillary-based DNA sequencing was studied. This base-stacking method allows direct injection of unpurified products of dye-primer sequencing reactions onto capillaries without any pretreatment. On-column concentration of DNA fragments is achieved simply by electrokinetic injection of hydroxide ions. A neutralization reaction between these OH- ions and the cationic buffer component Tris+ results in a zone of lower conductivity, within which field focusing occurs. DNA fragments are concentrated at the front of this low-conductivity zone. With sample injection times as long as 360 s at 50 V/cm, resolution could still be restored by the stacking process. Using a 36/47-cm-long uncoated capillary, with poly(dimethylacrylamide) as the separation matrix, and electric field of 160 V/cm, a resolution of at least 0.5 could be generated for fragments up to 650 nucleotides long. Both resolution and signal strength are excellent relative to conventional injection of highly purified samples. No significant degradation of the capillary performance was observed over at least 20 sequencing runs using this new sample injection methods.

Explore related subjects

Buffers DNA Electrophoresis, Capillary Hydrogen-Ion Concentration Sequence Analysis, DNA Journal Article Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Y Xiong, S R Park, H Swerdlow. 1998-09-01. Base stacking: pH-mediated on-column sample concentration for capillary DNA sequencing.. https://doi.org/10.1021/ac980376j

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2002

Liquid-liquid separation in solutions of normal and sickle cell hemoglobin.

We show that in solutions of human hemoglobin (Hb)--oxy- and deoxy-Hb A or S--of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40 degrees C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.

Buffers↗

PubMed2002

A novel ultraviolet assay for testing side reactions of carbodiimides.

Carbodiimides possess considerable absorbance in the ultraviolet region; the extinction coefficient of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in water is epsilon(214) = 6.3. 10(3) L x mol(-1) x cm(-1). It provides a very simple method for testing possible side reactions of carbodiimides. This technique was used to study effects of pH, different buffers, and other components typically present in biological samples on EDC stability. It was shown that the hydrolysis rate in pure water increased from 1.5. 10(-5) to 5. 10(-4) s(-1) by pH decreasing from 7 to 4. A strong increase of the rate of EDC loss was observed in the presence of different components. This effect can be described by the following row of hydrolysis rates: citrate >> acetate approximately phosphate >> SDS. The results can be used to optimize carbodiimide-mediated reactions of peptide bond formation in organic chemistry or peptide synthesis or during immobilization or cross-linking of biological molecules.

Buffers↗

PubMed2002

Hydrolytic degradation of nitrendipine and nisoldipine.

The development of a new HPLC-UV diode array procedure applied to follow the hydrolytic degradation of two well-known 4-nitrophenyl-1,4-dihydropyridine derivatives, nitrendipine and nisoldipine is reported. Hydrolysis of each drug were carried out in ethanol/Britton-Robinson buffer at different pHs, stored into amber vials at controlled temperatures of 40, 60 and 80 degrees C and periodically sampled and assayed by HPLC. Nitrendipine degradation in different parenteral solutions was also evaluated. The HPLC procedure exhibited an adequate selectivity, repeatability (<1%) and reproducibility (<2%). The recoveries were higher than 98% with CV of 1.13 and 1.54% for nitrendipine and nisoldipine, respectively. A significant degradation was observed at alkaline pH (>pH 8) with a first order kinetic for both drugs. At pH 12, 80 degrees C, k values of 3.56x10(-2) x h(-1) and 2.22x10(-2) for nitrendipine and nisoldipine, respectively were obtained. Also, activation energies of 16.8 and 14.7 kcal x mol(-1) for nitrendipine and nisoldipine, respectively, were calculated. Furthermore, from the results obtained from hydrolytic degradation in different solutions for parenteral use, we can affirm that solutions significantly increased the degradation of nitrendipine. In conclusion, the HPLC proposed procedure exhibited adequate analytical requirements to be applied to the hydrolytic degradation studies of nitrendipine and nisoldipine. Furthermore, all tested parenteral solutions significantly increased the hydrolytic degradation of nitrendipine, the composition of solution being a relevant factor.

Buffers↗