A Matrix for DNA Separation: Genotyping and Sequencing Using Poly(vinylpyrrolidone) Solution in Uncoated Capillaries

Abstract

We report a new sieving matrix for DNA separation based on commercially available poly(vinylpyrrolidone). The new sieving matrix has a very low viscosity at moderate concentrations, e.g., 27 cP at 4.5%. Its excellent self-coating property can reduce electroosmotic flow to a negligible level. Column regeneration between runs is very simple and effective. Successful separations were achieved in uncoated capillaries. For genotyping, we show that D1S80 and amelogenin sex determination system can be baseline separated as double-stranded DNA, and vWF, TH01, TPOX, and CSF1PO short tandem repeats can be separated with single-base resolution as single-stranded DNA in this new matrix. Sequencing of M13mp18 showed good resolution up to 500 bases in a solution of high-molecular-weight fraction extracted from commercially available PVP. The feasibility of adaptation to a multicapillary array system is discussed.