A Versatile Method for DNA Sequencing of Unpurified PCR Products using an Automated DNA Sequencer and Tailed or Nested Primer Labeled with Near-infrared Dye: A Case Study on the Harmful Dinoflagellate Alexandrium

摘要

DNA sequence-based typing is considered a robust tool for the discrimination of dinoflagellate species because of the availability of extensive rDNA sequences. Here, we present a rapid, cost-effective DNA-sequencing technique for various PCR products.This sequencing strategy relies on "nested" or "tailed" primer labeled with near-infrared dye, and uses a minimal volume of unpurified PCR product (ca. 5 mu L) as the DNA template for sequencing reactions. Reliable and accurate base identification was obtained for several hundred PCR fragments of rRNA genes. This quick, inexpensive technique is widely applicable to sequence-based typing in clinical applications, as well as to large-scale DNA sequencing of the same genomic regions from related species for studies of molecular evolution.