Method of translocating nucleic acids through nanopores

A method of analyzing a nucleic acid comprising:

• extending a primer on a template in a reaction mixture to produce a double stranded product comprising a labeled extended strand with a free 3′-hydroxyl;
• extending further the extended strand without a template with a terminal transferase activity to produce a 3′-single stranded tail on the double stranded product;
• providing at least one nanopore that separates and provides fluid communication between a first chamber and a second chamber, wherein each nanopore of the at least one nanopore is capable of passing a single stranded nucleic acid but not a double stranded nucleic acid;
• disposing double stranded product with the 3′single stranded tails in the first chamber;
• capturing a 3′ single stranded tail of a double stranded product by the at least one nanopore by applying an electrical field across the nanopore,
• translocating at a rate of less than 1000 nucleotides per second (nt/sec) the labeled extension strand of the captured double stranded product through the nanopore by the applied electrical field, wherein the translocating strand of the double stranded product is unzipped as it enters the nanopore.

A method of determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:

• providing a labeled double stranded products of target polynucleotides, wherein a labeled strand of each labeled double stranded product comprises a single stranded overhang and wherein different kinds of nucleotides of the labeled strand have different optical labels that generate distinct optical signals;
• providing at least one nanopore that provides fluid communication between a first chamber and a second chamber, wherein each nanopore is capable of passing a single stranded nucleic acid but not a double stranded nucleic acid;
• disposing the labeled double stranded product in the first chamber;
• capturing by the at least one nanopore a single stranded overhang of a labeled double stranded product by applying an electrical field across the nanopore;
• translocating the labeled stand through the nanopore so that the nucleotides of the labeled strand pass single file through an excitation zone at a rate of less than 1000 nucleotides per second (nt/sec), wherein optical labels are excited to generate optical signals and wherein the translocating labeled strand of the double stranded product is unzipped as it enters the nanopore;
• detecting a time series of optical signals from the optical labels as the labeled strand translocates through the nanopore to produce a strand optical signature;
• and
• determining a sequence of the target polynucleotide from the strand optical signature.