Molecular detection and counting using nanopores

Abstract

Provided herein are methods and compositions for detecting and/or quantitating target analytes, including nucleic acids and polypeptides, using nanopore detectable barcodes.

1 . A method for detecting and/or quantifying a target nucleic acid molecule, the method comprising:
 a. contacting the target nucleic acid molecule with at least one primer that specifically hybridizes to the target nucleic acid molecule, resulting in a target nucleic acid molecule-primer complex;   b. extending the primer using modified nucleotides, wherein the modified nucleotides comprise barcode units, to produce a target-modified nucleotide polymer complex;   c. isolating the modified nucleotide polymer;   d. detecting and/or quantifying the modified nucleotide polymer using a nanopore.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid molecule is selected from the group consisting of mRNA or cDNA. 
     
     
         3 . The method of any of  claim 2 , wherein the primer comprises oligo-dT. 
     
     
         4 . The method of  claim 2 , wherein the nucleic acid molecule is DNA. 
     
     
         5 . The method of  claim 1 , wherein the nucleic acid molecule is amplified. 
     
     
         6 . A method for detecting and/or quantifying a target nucleic acid molecule, the method comprising:
 a. contacting the target nucleic acid molecule with at least a first primer and a second primer, wherein:
 i. each primer comprises a target-specific region and a universal priming site; 
 ii. the second primer comprises barcode coding region disposed between its target-specific region and its universal priming site; 
   b. amplifying the target nucleic acid molecule to produce amplicons;   c. isolating and denaturing the amplicons to produce single-stranded amplicons;   d. contacting the single-stranded amplicons with the second primer;   e. extending the second primer using a modified nucleotides to produce an amplicon-modified nucleotide polymer complex;   f. isolating modified nucleotide polymer;   g. detecting and/or quantifying the modified nucleotide polymer using a nanopore.   
     
     
         7 . A multiplexed method for detecting and/or quantitating nucleic acid target molecules in a sample, comprising the steps of:
 a. Contacting sample with a set of at least two oligonucleotide primers, wherein at least one primer in each set comprises a barcode template, wherein each primer comprises a universal priming sequence, and wherein the oligonucleotide primers hybridize at adjacent sites on the target nucleic acid molecules;   b. Ligating the primers;   c. Amplifying the ligated nucleic acid molecules;   d. extending the second primer using a modified nucleotides to produce an amplicon-modified nucleotide polymer complex;   e. isolating modified nucleotide polymer;   f. detecting and/or quantifying the modified nucleotide polymer using a nanopore.   
     
     
         8 . The method of  claim 7 , wherein the barcode template comprises a sample index, a locus index, or a strand index. 
     
     
         9 . A method of detecting an analyte in a sample, comprising the steps of:
 a. Contacting the sample with a specific binding protein comprising a nanopore detectable barcode to produce an analyte-binding protein complex;   b. Isolating the analyte-binding protein complex;   c. Detecting the nanopore detectable barcode using a nanopore.   
     
     
         10 . The method of  claim 9 , wherein the specific binding protein is an antibody. 
     
     
         11 . The method of any  claim 9 , wherein the nanopore-detectable barcode is attached to the specific binding protein using click chemistry. 
     
     
         12 . The method of any  claim 10 , wherein the nanopore-detectable barcode is attached to the specific binding protein using click chemistry.