Nanopore Detection of Target Polynucleotides From Sample Background

A method for detecting the presence or absence of a target polynucleotide sequence suspected to be present in a sample, comprising:

• providing a set of primers, wherein at least one of said primers is hybridizable to a polynucleotide comprising said target polynucleotide sequence, and wherein at least one of said primers is bound to a payload molecule;
• performing an amplification reaction on said sample, wherein said sample comprises said primer set and reagents for amplification, such that an amplicon comprising said target polynucleotide sequence generated by said amplification reaction will be bound to said payload molecule;
• loading said sample into a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes, and configuring the device to pass the nucleic acid through one or more pores, wherein the device comprises a sensor for each pore that is configured to identify objects passing through the nanopore;
• and
• detecting the presence or absence of said target polynucleotide sequence in said sample by determining whether the target polynucleotide bound to the payload molecule passed through the nanopore using data from said sensor.

A method for detecting the presence or absence of a target polynucleotide sequence suspected to be present in a sample, comprising:

• providing a set of primers, wherein at least one of said primers is hybridizable to a polynucleotide comprising said target polynucleotide sequence;
• performing an amplification reaction on said sample, wherein said sample comprises said primer set and reagents for amplification, such that an amplicon comprising said target polynucleotide sequence generated by said amplification reaction is at least 100 base pairs in length;
• loading said sample into a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes, and configuring the device to pass the nucleic acid through one or more pores, wherein the device comprises a sensor for each pore that is configured to identify objects passing through the nanopore;
• and
• detecting the presence or absence of said target polynucleotide sequence in said amplified sample by determining whether the target polynucleotide bound to the payload molecule passed through the nanopore using data from said sensor, wherein said amplified sample has not been purified.

A kit comprising:

• a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes, and configuring the device to pass the nucleic acid through one or more pores, wherein the device comprises a sensor for each pore that is configured to identify objects passing through the nanopore;
• a primer set, wherein at least one of said primers is hybridizable to a polynucleotide comprising said target polynucleotide sequence, and wherein at least one of said primers is modified to comprise a conjugation site capable of specifically binding to a payload molecule;
• a payload molecule for binding to said conjugation site prior to, during, or after amplification;
• instructions for use to detect the presence or absence of said target polynucleotide in a sample.

A kit comprising:

• a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes, and configuring the device to pass the nucleic acid through one or more pores, wherein the device comprises a sensor for each pore that is configured to identify objects passing through the nanopore;
• a primer set, wherein at least one of said primers is hybridizable to a polynucleotide comprising said target polynucleotide sequence, wherein said primers generate an amplicon comprising said target polynucleotide sequence of at least 100, 200, 500, 1,000, 2,000, 5,000, or 10,000 base pairs during an amplification reaction;
• and
• instructions for use to detect the presence or absence of said target polynucleotide in a sample.

A method for quantifying the amount of target polynucleotide sequence present in a sample, comprising:

• providing a control sample comprising a known amount of a control polynucleotide and an experimental sample comprising an unknown amount of target polynucleotide;
• amplifying said control sample to generate a first amplicon comprising said control polynucleotide and amplifying said experimental sample to generate a second amplicon comprising said target polynucleotide;
• loading said control sample and said experimental sample separately into a device comprising a nanopore, wherein said nanopore separates an interior space of the device into two volumes, and configuring the device to pass the first or second amplicon through one or more pores, wherein the device comprises a sensor for each pore that is configured to identify objects passing through the nanopore;
• and
• comparing the capture rate of said first amplicon in the nanopore with the capture rate of said second amplicon in the nanopore to quantify the amount of target polynucleotide sequence in said experimental sample.

A method for quantifying the amount of target polynucleotide sequence present in a sample, comprising:

• providing a control sample comprising a known amount of a control polynucleotide and an experimental sample comprising an unknown amount of target polynucleotide;
• diluting said control sample to generate at least two different known concentrations of said control polynucleotide;
• loading said control sample into a device comprising a nanopore at said at least two different known concentrations of said control polynucleotide, wherein said nanopore separates an interior space of the device into two volumes, and configuring the device to pass the control polynucleotide through one or more pores, wherein the device comprises a sensor for each pore that is configured to identify objects passing through the nanopore;
• detecting the capture rate in said nanopore of the control polynucleotide at each of said at least two known concentrations;
• amplifying said experimental sample to generate an amplicon comprising said target polynucleotide;
• and
• loading the experimental sample into said device comprising a nanopore;
• detecting the capture rate of the target polynucleotide in said nanopore;
• comparing the capture rate of said first amplicon in the nanopore with the capture rate of said second amplicon in the nanopore to quantify the amount of target polynucleotide sequence in said experimental sample.