Temperature-independent Detection of Heteroduplex and Homoduplex Fragments Applying
Abstract:
Monoliths of poly(glycidylmethacrylate-co-divinylbenzene) were prepared in the confines of presilanized borosilicate glass columns (100 × 3 mm I.D.). These monoliths were surface modified into strong anion-exchangers with hydrochloric acid (10%) and triethylamine, successively. The strong anion-exchanger established good separation of 5-phosphorylated oligodeoxythymidylic acids fragments [d(pT)(12-18)]. Moreover, heteroduplex and homoduplex fragments of a low-range mutation standard [of STS marker from the Y-chromosome (209 bp)] were separated at ambient and elevated temperatures using sodium phosphate buffer and a gradient former of sodium chloride, in anion-exchange high-performance liquid chromatography (AE-HPLC). This is a step forward for mutation detection as temperature-independent method, which is not the case in denatured ion-paired reversed-phase chromatography (D-IP-RP-HPLC), where mutation detection is temperature critical and might be bypassed if temperature changes slightly. Finally, reproducibility check from run-to-run and monolith-to-monolith showed a relative standard deviation (RSD) of less than 2%.
