Sample stacking during membrane-mediated loading in automated DNA sequencing.
Microporous membrane-mediated loading is a novel and efficient sample injection technique for ultrathin slab gel electrophoresis-based automated DNA sequence analysis. The sequencing reaction mixture is spotted directly onto the tabs of the membrane loader, which is then inserted to close proximity of the straight edge of the separation gel. The use of a higher viscosity (> 60 cSt), low ionic strength (conductivity < 320 microS) well solution with a pH of more than one unit lower that of the separation and gel buffer system makes possible efficient stacking of the DNA sequencing fragments at the interface of the gel matrix. When the injection/separation process is initiated by the application of the electric field, a high local electric potential drop is formed across the low ionic strength well solution zone. This results in very fast migration of the DNA sequencing fragments toward the interface of the separation gel, where they stack up against the higher conductivity sieving matrix. During this stacking process, primarily the local pH mediates the actual mobility of the buffer co-ions (borate), forming the leading and terminating zones in the well solution and separation gel, respectively.