A general method for routine sequencing of cloned DNA fragments using commercial dye primers and its application in sequence analysis of Toxoplasma gondii RH genome fragments

Abstract

A generally applicable approach for amplification and subsequent sequencing using commercially available dye primers is described. The polymerase chain reaction primers, one of which is biotinylated, are tagged at their 5' end with sequences of commercial sequencing primers. After purification and strand separation on streptavidin-coated magnetic beads, both strands are sequenced automatically on an Applied Biosystems 373A DNA sequencer using the appropriate standard dye-labeled sequencing primers. This approach is demonstrated on PstI and MnlI DNA fragments from Toxoplasma gondii RH cloned in the in-house developed derivatives of vector pJRtac99.

Key Words:

• automated DNA sequencing
• magnetic separation
• PCR