Characterization of a microdialysis approach to prepare polymerase chain reaction products for electrospray ionization mass spectrometry using on-line ultraviolet absorbance measurements and inductively coupled plasma-atomic emission spectroscopy

Abstract

The desalting efficiencies for microdialysis have been quantitatively determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES) by analyzing samples which mimic polymerase chain reaction (PCR) conditions. Desalting efficiencies for the removal of Mg2+ from samples containing oligonucleotides were typically 99.94% for a single dialysis experiment and 99.98% for a tandem-dialysis experiment. The determination of the molecular weight selectivity of a microdialysis fiber with a 13,000 Da molecular weight cutoff (MWCO) employing a 10 mM NH4OAc countercurrent buffer was accomplished using on-line ultraviolet (UV) absorbance. Results revealed an insensitivity to molecular weight over a broad range represented by dNTPs (ca. 490 Da), PCR primers (ca. 5100 Da), and PCR products (extending to ca. 100 kDa) thus showing the inability of microdialysis to remove unincorporated deoxyribonucleotide 5′-triphosphates (dNTPs) and primers from PCRs. A comparison study conducted utilizing proteins ranging from ca. 1 to 29 kDa produced recoveries dictated by the MWCO of the microdialysis fiber. Recoveries were zero for the smallest proteins tested (ca. 1.3 and 2.9 kDa), but increased to 98% for the largest protein (ca. 29 kDa). The ability of microdialysis to convert double-stranded DNA to single-stranded DNA due to a rapid decrease in the ionic strength was examined along with the effects of buffer concentration, temperature, pH, tandem dialysis, and a denaturing additive, formamide. Melting temperature curves showed that microdialyzed samples remain double stranded while utilizing NH4OAc buffer concentrations of 0 (i.e. pure water) to 10 mM. Adjustment of the buffer up to pH 10.98, temperature increases to 51 °C, tandem dialysis, and the addition of formamide were also unable to produce the conversion of ds-DNA to ss-DNA in detectable amounts. Copyright © 1999 John Wiley & Sons, Ltd.