In large DNA sequencing projects, the largest fraction of the costs, such as synthesis of oligonucleotides and skilled tasks such as performing reactions and pouring and running gels, are incurred on a per-sequencing-reaction basis. We will apply the Constrained Uniform Field Electrophoresis (CUFE) system to extract more information from each sequencing reaction, thereby greatly reducing the cost of sequencing. Previous studies have shown that separation of single- stranded DNA molecules can be enhanced by pulsed-field techniques. CUFE is a type of pulsed-field electrophoresis invented by MJ Research. A number of features of CUFE make it uniquely suitable for application to sequencing gels. We will first construct a CUFE apparatus designed specifically for polyacrylamide sequencing gels. We will then test and modify the apparatus to optimize lane straightness and other gel parameters. Finally we will determine the extent to which pulsed-field electrophoresis is capable of increasing separation of single-stranded DNA fragments of 300 bases and higher in length.
