Discrete mobility of single-stranded DNA in non-denaturing gel electrophoresis

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Oxford University Press

RESUMO

Gel electrophoresis is the standard method to separate, identify and purify nucleic acids. SSCP detects single base changes by altered mobility of single-stranded segments electrophoresed through non-denaturing polyacrylamide gels. Herein, changes in electrophoretic mobilities due to single base substitutions were measured for single-stranded segments of lengths ranging from 333 to 547 nt. A 484 nt segment in exon H of the human factor IX gene was studied most intensively. After SSCP, mobilities were determined by scanning autoradiograms at very high resolution (1200 d.p.i.), which allowed precise measurement of mobilities. When the mobilities of 46 single base substitutions were characterized, the distribution of mutant segments relative to a wild-type control was found to be discrete, i.e. the observed mobility values occurred in distinct ranges. Discrete mobility distributions were seen at different electrophoretic temperatures, buffer concentrations, segment lengths and segment sequences. In addition: (i) single base substitutions caused discontinuous distributions between highly dispersed and sharp bands; (ii) at least one single-stranded segment produced two sharp bands of similar intensity. These observations suggest that: (i) the single base changes in DNA segments in the size range 333–547 nt result in discrete conformational changes; (ii) individual DNA molecules of the same DNA segment can occasionally adopt two or more discrete conformations.

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