New protocols for DNA sequencing with dye terminators

Abstract

We have developed a new two-step protocol for Taq cycle sequencing using the ABI dye terminators. First, linear PCR is used to extend a single sequencing primer in the absence of dye terminators. This nested set of polynucleotides as well as the original primer then serve as starting points for the cycled termination reactions. Using this method it is easily possible to generate dye terminator raw data with much improved signal intensity beyond 400 to 500 bp. We also present a quick and convenient method to remove excess of dye terminators by gel filtration using 24 mini columns fixed in a block of perspex.

Key Words:

• Taq cycle sequencing
• dye terminators
• multiple primers
• parallel gel filtration
• mini columns