Evaluation of sieving polymers for fast, reproducible electrophoretic analysis of short tandem repeats (STR) in capillaries.
Efficient capillary electrophoretic STR analysis requires rapid, reproducible and robust separation of DNA fragments with reasonable capillary longevity--this is currently accomplished using proprietary commercial polymeric sieving matrices specifically developed for this separation. These matrices, while effective, are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths, increasing the time required to accomplish the separation and minimizing the potential extrapolation to other miniaturized platforms. As the forensic community looks toward next generation microchip technology as a means of processing casework more rapidly, new sieving polymers need to be evaluated for utilization in this platform. The research presented here describes the assessment of commercially-available polymeric sieving matrices for STR analysis, with consideration given to feasibility of incorporation into a microdevice. Polymer composition, molecular weight, and concentration were evaluated, along with an assessment of the effects of buffer composition, separation temperature, and capillary length. These variables were evaluated individually or collectively on the ability to resolve STR DNA fragments and the reproducibility of the separations and the results compared to a proprietary commercial product. A 600,000 Da MW poly(ethylene oxide) (PEO) solution at a 3% (w/v) concentration was determined to be the most suitable matrix for these separations. This polymer, in coated capillaries, provided highly robust and reproducible separations, with near baseline resolution of fragments having single base differences. Reductions in the temperature of the separation, from 60 degrees C to 40 degrees C, and the urea concentration of the buffer, from 7 M to 3.5 M, provided increased longevity of the PEO polymer for repeated separations. Comparison of this polymer with currently specified commercial products used for STR analysis showed that the optimized PEO matrix provided superior separations under all conditions tested. In addition, PEO could be utilized in shorter capillary systems, with a concurrent decrease in analysis time, highlighting its potential for use in shortened capillary or microdevice systems.
Bienvenue
Joan M
JM
Department of Chemistry, McCormick Road, University of Virginia, Charlottesville, VA 22904, USA.
Wilson
Kate L
KL
Landers
James P
JP
Ferrance
Jerome P
JP
D002021:Buffers; D004247:DNA / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D005240:Feasibility Studies; D006801:Humans; D011092:Polyethylene Glycols / Q000737:chemistry*; D013501:Surface-Active Agents / Q000737:chemistry*; D020080:Tandem Repeat Sequences; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D002021:Buffers; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D014508:Urea; D004247:DNA