Simultaneous Bi-Directional Cycle Sequencing.

Abstract

A new method termed bi-directional cycle sequencing is reported in which both strands of duplex DNA (plasmid or PCR product) are sequenced simultaneously by combining a forward and reverse primer (each labeled with a different fluorophore) in the same sequencing reaction. The LI-COR IR² Automated DNA Sequencing System is used to detect and analyze both the forward and reverse sequences of this bi-directional reaction in parallel.

Sequencing double-stranded DNA bi-directionally has major advantages over traditional cycle sequencing methods. First, detection sensitivity is increased for smaller inserts and PCR products (< 1.5 kb). Some primer extensions will reach to the opposite priming site, resulting in amplification of the original amount of DNA template. Second, the ends of vector inserts are clearly delineated as if sequencing a PCR product (useful during sequence editing). Third, bi-directional cycle sequencing accommodates the new possibility of sequentially amplifying and sequencing directly from single colonies without the intermediate purification steps. Fourth, since confirming sequence is generated in the same reaction, it is easier to resolve ambiguities in one strand using the sequence of the complementary strand. Finally, time and reagent consumption are halved by combining the two reactions.