ACTIVATION-ENERGY OF SINGLE-STRANDED-DNA MOVING THROUGH CROSS-LINKED POLYACRYLAMIDE GELS AT 300-V CM - EFFECT OF TEMPERATURE ON SEQUENCING RATE IN HIGH-ELECTRIC-FIELD CAPILLARY GEL-ELECTROPHORESIS/

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a f unction of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compa red with the predictions of the biased reptation model. The model pred icts that the fragment length for the onset of biased reptation with s tretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperat ure. Biased reptation fails to model accurately the temperature depend ence of mobility. We analyzed the data and extracted the activation en ergy for passage of sequencing fragments through the gel. For fragment s containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/m ol per base. This transition in the activation energy presumably refle cts a change in conformation of the DNA fragments; small fragments exi st in a random coil configuration and larger fragments migrate in an e longated configuration.