Fast DNA sequencing up to 1000 bases by capillary electrophoresis using poly(N,N-dimethylacrylamide) as a separation medium

Poly(N,N-dimethylacrylamide) (PDMA) with a molecular mass of 5.2 x 10(6) g/ mol has been synthesized and used in DNA sequencing analysis by capillary e lectrophoresis (CE). A systematic investigation is presented on the effects of different separation conditions, such as injection amount, capillary in ner diameter, polymer concentration, effective separation length, electric field and temperature, on the resolution. DNA sequencing up to 800 bases wi th a resolution (R) limit of 0.5 (and 1000 bases with a resolution limit of 0.3) and a migration time of 96 min was achieved by using 2.5% w/v polymer , 150 V/cm separation electric field, and 60 cm effective separation length at room temperature on a DNA sample prepared with FAM-labeled -21M13 forwa rd primer on pGEM3Zf(+) and terminated with ddCTP. Ultrafast acid fast DNA sequencing up to 420 and 590 bases (R greater than or equal to 0.5) were al so achieved by using 3% w/v polymer and 40 cm effective separation length w ith a separation electric field of 525 and 300 V/cm, and a migration time o f 12.5 and 31.5 min, respectively. PDMA has low viscosity, long shelf life and dynamic coating ability to the glass surface. The unique properties of PDMA make it a very good candidate as a separation medium for large-scale D NA sequencing by capillary array electrophoresis (CAE).