DNA sequencing using ultra small amounts of reagents and template

Abstract

One of the key points in the genome project is finding ways to reduce the running cost in DNA sequencing. One way is to use a highly-sensitive fluorescent DNA sequencer, where only trace amounts of template DNA and reagents are needed. An experimental protocol optimized for the trace amounts of DNA analysis was established by using the hybridization reaction rate coefficient of primers on template DNA, which was estimated to be 7.5×105 M-1sec-1 at 37°C. One femtomole of template DNA with 0.001 unit of modified T7 DNA polymerase (Sequenase Ver. 2.0) and also 0.45 fmol of M13 template DNA with 0.01 unit of Taq DNA polymerase were enough to sequence DNA of up to 400 bases.