Method of sequencing multiple copies of a sequence in a circular template

Abstract

The invention relates to methods for nanopore sequencing. A substrate such as a semiconductor is provided having a nanopore or an array of nanopores connecting an upper solution with a lower solution. A polymerase-nucleic acid complex comprising a strand displacing DNA polymerase and a circular nucleic acid template and components required for DNA synthesis are provided. The polymerase produces a nascent DNA strand complementary to the circular nucleic acid template and this nascent strand is translocated through the nanopore while it is being produced by a polymerase-nucleic acid complex comprising a strand displacing DNA polymerase and a circular nucleic acid template and providing the components required for DNA synthesis whereby the polymerase produces a nascent DNA strand which is translocated through the nanopore while the nascent DNA strand is being produced. The sequence of the nascent DNA strand is determined while it translates through the pore using the measured current over time. The polymerase proceeds around the circular template and continues to produce the nascent DNA strand such that a sequence in the nascent strand corresponding a sequence in the circular template is determined more than once. Determining the sequence of corresponding to the circular template more than once can provide for greater accuracy.